Biological materials related to CXCR7

ABSTRACT

The present invention relates to particular polypeptides, nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions and in particular to pharmaceutical compositions that comprise such polypeptides, for prophylactic, therapeutic or diagnostic purposes.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application No. 61/318,000, filed Mar. 26, 2010, thedisclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to biological materials related to CXCR7and more in particular to polypeptides, nucleic acids encoding suchpolypeptides; to methods for preparing such polypeptides; to host cellsexpressing or capable of expressing such polypeptides; to compositionsand in particular to pharmaceutical compositions that comprise suchpolypeptides, for prophylactic, therapeutic or diagnostic purposes.

BACKGROUND OF THE INVENTION

Although it is suggested in the art i) that the blockage of CXCR7employed along with CXCR4 blockage may be useful for the treatment ofSDF-1-dependent tumor progression and metastasis (R B Maksym et al.,2009, The role of stromal-derived factor-1-CXCR7 axis in development ofcancer, European Journal of Pharmacology, 625 (1-3), pages 31-40) andii) that some small molecular inhibitors, such as CCX733 or CCX266,siRNA and blocking antibodies (clones 11G8, 9C4 see e.g. US20070167443;clone 358426 (R&D Systems); 8F11 (Biolegend)), may be useful fortherapeutic interference with CXCR4-mediated activation of integrins (TN Hartmann et al., 2008, A crosstalk between intracellular CXCR7 andCXCR4 involved in rapid CXCL12-triggered integrin activation but not inchemokine-triggered motility of human T lymphocytes and CD34+ cells,Journal of Leukocyte Biology, 84, pages 11301140), the biology of CXCR7is still poorly understood since the mechanism(s) of action throughwhich CXCR7 acts is unclear since i) it may act as a kind of decoy orsignalling receptor depending on cell type—R M Maksym et al., supra andsince ii) the interplay between ITAC and SDF-1 binding to CXCR7 isunclear.

The identification of selective therapeutically effective anti-CXCR7agents is not only challenging because of its poorly understood biology(such as e.g. mechanism of action e.g. of the potential agonists CCX733or CCX266 versus antagonists, interplay with CXCR4, recognition ofimportant epitopes, cross-reactivity of the compounds CCX733 or CCX266and associated toxicity), it is also acknowledged in the art (see e.g.Naunyn-Schmied Archives Pharmacology 379: 385-388) that the generationof an anti-GPCR therapeutic agent such as an anti-CXCR7 agent isdifficult since i) the native conformation of active CXCR7 in cancercells is not exactly known, since ii) it is expected that CXCR7 showslow immunogenicity (due to a limited number of extracellular surfaceexposed amino acid residues that are in addition very conserved, e.g.mouse-human CXCR7 is 96% homologues).

Furthermore, compounds (CCX733, CCX754) selectively blocking binding ofCXCL11 and CXCL12 to CXCR7 function like chemokine ligands with respectto homodimerization, i.e. enhance CXCR7 homodimerization by 2.5 to 3.5fold with significant increases (P<0.05) first detected at 10 and 100 nM(K E Luker et al., 2009, Imaging chemokine receptor dimerization withfirefly luciferase complementation, FASEB journal, 23, pages 823-834).

Targeting serum albumin to extend the half-life of biological moleculessuch as e.g. immunoglobulin single variable domains has been describede.g. in WO2008/028977.

The generation of a conventional anti-CXCR7 antibody has been describede.g. in WO2006116319 for conventional antibodies 11G8, 6E10 and in Zabelet al. for conventional antibody 8F11 (Zabel et al., 2009, Elucidationof CXCR7 mediated signalling events and inhibition of CXCR4 mediatedtumor cell transendothelial migration by CXCR7 ligands. J. Immunol.; 183(5):3204-11). However, it is unclear at present whether these or similarantibodies are suitable for a medical application

SUMMARY OF THE INVENTION

The art is in need of potent anti-CXCR7 agents that can credibly exploreand establish the medical potential of this target and, furthermore, isin need of diagnostically, preventatively, and/or therapeuticallysuitable anti-CXCR7 agents such as provided herein.

Immunoglobulin sequences, such as antibodies and antigen bindingfragments derived therefrom (e.g. immunoglobulin single variabledomains) are used to specifically target their respective antigens inresearch and therapeutic applications. The generation of immunoglobulinsingle variable domains such as e.g. VHHs may involve the immunizationof an experimental animal such as a Llama, construction of phagelibraries from immune tissue, selection of phage displaying antigenbinding immunoglobulin single variable domains and screening of saiddomains and engineered constructs thereof for the desired specificities(WO 94/04678). Alternatively, similar immunoglobulin single variabledomains such as e.g. dAbs can be generated by selecting phage displayingantigen binding immunoglobulin single variable domains directly fromnaïve or synthetic libraries and subsequent screening of said domainsand engineered constructs thereof for the desired specificities (Ward etal, Binding activities of a repertoire of single immunoglobulin variabledomains secreted from Escherichia coli, Nature, 1989, Oct. 12; 341(6242): 544-6); Holt et al., Trends Biotechnol., 2003, 21(11):484-490;as well as for example WO 06/030220, WO 06/003388 and other publishedpatent applications of Domantis Ltd.).

The present invention relates to particular polypeptides that compriseor more preferably essentially consist of i) a first building blockconsisting essentially of one or more (preferably one) immunoglobulinsingle variable domain(s), wherein said immunoglobulin single variabledomain(s) is(are) directed against CXCR7 and in particular against humanCXCR7; and ii) a second building block consisting essentially of one ormore (preferably one) immunoglobulin single variable domain(s), whereinsaid immunoglobulin single variable domain(s) is(are) directed againstserum albumin and in particular against human serum albumin (and evenmore preferably wherein said immunoglobulin single variable domain isAlb8 (as herein defined)). Furthermore, the invention also relates tonucleic acids encoding such polypeptides; to methods for preparing suchpolypeptides; to host cells expressing or capable of expressing suchpolypeptides; to compositions and in particular to pharmaceuticalcompositions that comprise such polypeptides, nucleic acids and/or hostcells; and to uses of such polypeptides, nucleic acids, host cellsand/or compositions for prophylactic, therapeutic or diagnosticpurposes. Other aspects, embodiments, advantages and applications of theinvention will become clear from the further description herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are illustrative only and are not required for enablement ofthe invention disclosed herein.

FIG. 1 shows an SDF1 competition experiment using FACS.

FIG. 2 shows an 11G8 competition experiment using FACS.

DESCRIPTION OF THE INVENTION

Definitions:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks mentioned in    paragraph a) on page 46 of WO 08/020079.-   b) Unless indicated otherwise, the term “immunoglobulin single    variable domain” is used as a general term to include but not    limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H) or V_(L) domains, respectively. The terms    antigen-binding molecules or antigen-binding protein are used    interchangeably and include also the term Nanobodies. The    immunoglobulin single variable domains further are light chain    variable domain sequences (e.g. a V_(L)-sequence), or heavy chain    variable domain sequences (e.g. a V_(H)-sequence); more    specifically, they can be heavy chain variable domain sequences that    are derived from a conventional four-chain antibody or heavy chain    variable domain sequences that are derived from a heavy chain    antibody. Accordingly, the immunoglobulin single variable domains    can be domain antibodies, or immunoglobulin sequences that are    suitable for use as domain antibodies, single domain antibodies, or    immunoglobulin sequences that are suitable for use as single domain    antibodies, “dAbs”, or immunoglobulin sequences that are suitable    for use as dAbs, or Nanobodies, including but not limited to V_(HH)    sequences. The invention includes immunoglobulin sequences of    different origin, comprising mouse, rat, rabbit, donkey, human and    camelid immunoglobulin sequences. The immunoglobulin single variable    domain includes fully human, humanized, otherwise sequence optimized    or chimeric immunoglobulin sequences. The immunoglobulin single    variable domain and structure of an immunoglobulin single variable    domain can be considered—without however being limited thereto—to be    comprised of four framework regions or “FR's”, which are referred to    in the art and herein as “Framework region 1” or “FR1”; as    “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and    as “Framework region 4” or “FR4”, respectively; which framework    regions are interrupted by three complementary determining regions    or “CDR's”, which are referred to in the art as “Complementarity    Determining Region 1” or “CDR1”; as “Complementarity Determining    Region 2” or “CDR2”; and as “Complementarity Determining Region 3”    or “CDR3”, respectively. It is noted that the terms Nanobody or    Nanobodies are registered trademarks of Ablynx N.V. and thus may    also be referred to as Nanobody® and/or Nanobodies®).-   c) Unless indicated otherwise, the terms “immunoglobulin sequence”,    “sequence”, “nucleotide sequence” and “nucleic acid” are as    described in paragraph b) on page 46 of WO 08/020079. The term    Nanobody is also as defined in WO 08/020079, and as described    therein generally refers to an immunoglobulin heavy chain variable    domain that has the functional and/or structural characteristics of    a V_(HH) domain (e.g. a V_(H) domain from the “heavy-chain only”    antibodies that occur in Camelids), and as such may in particular be    a (native) V_(HH), a humanized V_(HH) or a camelized V_(H), such as    a camelized human V_(H).-   d) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   e) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code. Reference is made to    Table A-2 on page 48 of the International application WO 08/020079    of Ablynx N.V. entitled “Immunoglobulin single variable domains    directed against IL-6R and polypeptides comprising the same for the    treatment of diseases and disorders associated with 11-6 mediated    signalling”.-   f) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated or    determined as described in paragraph e) on page 49 of WO 08/020079    (incorporated herein by reference), such as by dividing [the number    of nucleotides in the first nucleotide sequence that are identical    to the nucleotides at the corresponding positions in the second    nucleotide sequence] by [the total number of nucleotides in the    first nucleotide sequence] and multiplying by [100%], in which each    deletion, insertion, substitution or addition of a nucleotide in the    second nucleotide sequence—compared to the first nucleotide    sequence—is considered as a difference at a single nucleotide    (position); or using a suitable computer algorithm or technique,    again as described in paragraph e) on pages 49 of WO 08/020079    (incorporated herein by reference).-   g) For the purposes of comparing two or more immunoglobulin single    variable domains or other amino acid sequences such e.g. the    polypeptides of the invention etc, the percentage of “sequence    identity” between a first amino acid sequence and a second amino    acid sequence (also referred to herein as “amino acid identity”) may    be calculated or determined as described in paragraph f) on pages 49    and 50 of WO 08/020079 (incorporated herein by reference), such as    by dividing [the number of amino acid residues in the first amino    acid sequence that are identical to the amino acid residues at the    corresponding positions in the second amino acid sequence] by [the    total number of amino acid residues in the first amino acid    sequence] and multiplying by [100%], in which each deletion,    insertion, substitution or addition of an amino acid residue in the    second amino acid sequence—compared to the first amino acid    sequence—is considered as a difference at a single amino acid    residue (position), i.e. as an “amino acid difference” as defined    herein; or using a suitable computer algorithm or technique, again    as described in paragraph f) on pages 49 and 50 of WO 08/020079    (incorporated herein by reference).    -   Also, in determining the degree of sequence identity between two        immunoglobulin single variable domains, the skilled person may        take into account so-called “conservative” amino acid        substitutions, as described on page 50 of WO 08/020079. Any        amino acid substitutions applied to the polypeptides described        herein may also be based on the analysis of the frequencies of        amino acid variations between homologous proteins of different        species developed by Schulz et al., Principles of Protein        Structure, Springer-Verlag, 1978, on the analyses of structure        forming potentials developed by Chou and Fasman, Biochemistry        13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978, and on the        analysis of hydrophobicity patterns in proteins developed by        Eisenberg et al., Proc. Natl. Acad. Sci. USA 81: 140-144, 1984;        Kyte & Doolittle; J. Molec. Biol. 157: 105-132, 198 1, and        Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986, all        incorporated herein in their entirety by reference. Information        on the primary, secondary and tertiary structure of Nanobodies        is given in the description herein and in the general background        art cited above. Also, for this purpose, the crystal structure        of a V_(HH) domain from a llama is for example given by Desmyter        et al., Nature Structural Biology, Vol. 3, 9, 803 (1996);        Spinelli et al., Natural Structural Biology (1996); 3, 752-757;        and Decanniere et al., Structure, Vol. 7, 4, 361 (1999). Further        information about some of the amino acid residues that in        conventional V_(H) domains form the V_(H)/V_(L) interface and        potential camelizing substitutions on these positions can be        found in the prior art cited above.-   h) Immunoglobulin single variable domains and nucleic acid sequences    are said to be “exactly the same” if they have 100% sequence    identity (as defined herein) over their entire length.-   i) When comparing two immunoglobulin single variable domains, the    term “amino acid difference” refers to an insertion, deletion or    substitution of a single amino acid residue on a position of the    first sequence, compared to the second sequence; it being understood    that two immunoglobulin single variable domains can contain one, two    or more such amino acid differences.-   j) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this has the meaning given in    paragraph i) on pages 51-52 of WO 08/020079.-   k) The term “in essentially isolated form” has the meaning given to    it in paragraph j) on pages 52 and 53 of WO 08/020079.-   l) The terms “domain” and “binding domain” have the meanings given    to it in paragraph k) on page 53 of WO 08/020079.-   m) The terms “antigenic determinant” and “epitope”, which may also    be used interchangeably herein, have the meanings given to it in    paragraph 1) on page 53 of WO 08/020079.-   n) As further described in paragraph m) on page 53 of WO 08/020079,    an amino acid sequence (such as an antibody, a polypeptide of the    invention, or generally an antigen binding protein or polypeptide or    a fragment thereof) that can (specifically) bind to, that has    affinity for and/or that has specificity for a specific antigenic    determinant, epitope, antigen or protein (or for at least one part,    fragment or epitope thereof) is said to be “against” or “directed    against” said antigenic determinant, epitope, antigen or protein.-   o) The term “specificity” has the meaning given to it in    paragraph n) on pages 53-56 of WO 08/020079; and as mentioned    therein refers to the number of different types of antigens or    antigenic determinants to which a particular antigen-binding    molecule or antigen-binding protein (such as a polypeptide of the    invention) molecule can bind. The specificity of an antigen-binding    protein can be determined based on affinity and/or avidity, as    described on pages 53-56 of WO 08/020079 (incorporated herein by    reference), which also describes some preferred techniques for    measuring binding between an antigen-binding molecule (such as a    polypeptide of the invention) and the pertinent antigen. Typically,    antigen-binding proteins (such as the immunoglobulin single variable    domains, and/or polypeptides of the invention) will bind to their    antigen with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²    moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or    less and more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an    association constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and    preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸    to 10¹² liter/moles). Any K_(D) value greater than 10⁴ mol/liter (or    any K_(A) value lower than 10⁴ M⁻¹) liters/mol is generally    considered to indicate non-specific binding. Preferably, a    monovalent immunoglobulin single variable domain of the invention    will bind to the desired antigen with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM. Specific binding of an antigen-binding protein    to an antigen or antigenic determinant can be determined in any    suitable manner known per se, including, for example, Scatchard    analysis and/or competitive binding assays, such as    radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich    competition assays, and the different variants thereof known per se    in the art; as well as the other techniques mentioned herein. As    will be clear to the skilled person, and as described on pages 53-56    of WO 08/020079, the dissociation constant may be the actual or    apparent dissociation constant. Methods for determining the    dissociation constant will be clear to the skilled person, and for    example include the techniques mentioned on pages 53-56 of WO    08/020079.-   p) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as described in    paragraph o) on page 57 of WO 08/020079 and as mentioned therein    refers to the time taken for the serum concentration of the amino    acid sequence, compound or polypeptide to be reduced by 50%, in    vivo, for example due to degradation of the sequence or compound    and/or clearance or sequestration of the sequence or compound by    natural mechanisms. The in vivo half-life of an amino acid sequence,    compound or polypeptide of the invention can be determined in any    manner known per se, such as by pharmacokinetic analysis. Suitable    techniques will be clear to the person skilled in the art, and may    for example generally be as described in paragraph o) on page 57 of    WO 08/020079. As also mentioned in paragraph o) on page 57 of WO    08/020079, the half-life can be expressed using parameters such as    the t1/2-alpha, t1/2-beta and the area under the curve (AUC).    Reference is for example made to the Experimental Part below, as    well as to the standard handbooks, such as Kenneth, A et al:    Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists    and Peters et al, Pharmacokinete analysis: A Practical Approach    (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D    Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The    terms “increase in half-life” or “increased half-life” as also as    defined in paragraph o) on page 57 of WO 08/020079 and in particular    refer to an increase in the t1/2-beta, either with or without an    increase in the t1/2-alpha and/or the AUC or both.-   q) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization) of the target or antigen; or    any other site, epitope, antigenic determinant, part, domain or    stretch of amino acid residues on the target or antigen that is    involved in a biological action or mechanism of the target or    antigen. More generally, an “interaction site” can be any site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen to which an amino acid    sequence or polypeptide of the invention can bind such that the    target or antigen (and/or any pathway, interaction, signalling,    biological mechanism or biological effect in which the target or    antigen is involved) is modulated (as defined herein).-   r) An immunoglobulin single variable domain or polypeptide is said    to be “specific for” a first target or antigen compared to a second    target or antigen when is binds to the first antigen with an    affinity/avidity (as described above, and suitably expressed as a    K_(D) value, K_(A) value, K_(off) rate and/or K_(on) rate) that is    at least 10 times, such as at least 100 times, and preferably at    least 1000 times, and up to 10.000 times or more better than the    affinity with which said amino acid sequence or polypeptide binds to    the second target or polypeptide. For example, the first antigen may    bind to the target or antigen with a K_(D) value that is at least 10    times less, such as at least 100 times less, and preferably at least    1000 times less, such as 10.000 times less or even less than that,    than the K_(D) with which said amino acid sequence or polypeptide    binds to the second target or polypeptide. Preferably, when an    immunoglobulin single variable domain or polypeptide is “specific    for” a first target or antigen compared to a second target or    antigen, it is directed against (as defined herein) said first    target or antigen, but not directed against said second target or    antigen.-   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an immunoglobulin    single variable domain or polypeptide to interfere with the binding    directly or indirectly through allosteric modulation of other    immunoglobulin single variable domains or polypeptides of the    invention to a given target. The extend to which an immunoglobulin    single variable domain or polypeptide of the invention is able to    interfere with the binding of another to target, and therefore    whether it can be said to cross-block according to the invention,    can be determined using competition binding assays. One particularly    suitable quantitative cross-blocking assay uses a FACS- or an    ELISA-based approach to measure competition between the labelled    (e.g. His tagged or radioactive labelled) immunoglobulin single    variable domain or polypeptide according to the invention and the    other binding agent in terms of their binding to the target. The    experimental part generally describes suitable FACS-, ELISA- or    radioligand-displacement-based assays for determining whether a    binding molecule cross-blocks or is capable of cross-blocking an    immunoglobulin single variable domain or polypeptide according to    the invention. It will be appreciated that the assay can be used    with any of the immunoglobulin single variable domains or other    binding agents described herein. Thus, in general, a cross-blocking    amino acid sequence or other binding agent according to the    invention is for example one which will bind to the target in the    above cross-blocking assay such that, during the assay and in the    presence of a second amino acid sequence or other binding agent of    the invention, the recorded displacement of the immunoglobulin    single variable domain or polypeptide according to the invention is    between 60% and 100% (e.g. in ELISA/radioligand based competition    assay) or between 80% to 100% (e.g. in FACS based competition assay)    of the maximum theoretical displacement (e.g. displacement by cold    (e.g. unlabeled) immunoglobulin single variable domain or    polypeptide that needs to be cross-blocked) by the to be tested    potentially cross-blocking agent that is present in an amount of    0.01 mM or less (cross-blocking agent may be another conventional    monoclonal antibody such as IgG, classic monovalent antibody    fragments (Fab, scFv)) and engineered variants (diabodies,    triabodies, minibodies, VHHs, dAbs, VHs, VLs).-   t) An amino acid sequence such as e.g. an immunoglobulin single    variable domain or polypeptide according to the invention is said to    be “cross-reactive” for two different antigens or antigenic    determinants (such as serum albumin from two different species of    mammal, such as human serum albumin and cyno serum albumin) if it is    specific for (as defined herein) both these different antigens or    antigenic determinants.-   u) As further described in paragraph q) on pages 58 and 59 of WO    08/020079 (incorporated herein by reference), the amino acid    residues of an immunoglobulin single variable domain are numbered    according to the general numbering for V_(H) domains given by Kabat    et al. (“Sequence of proteins of immunological interest”, US Public    Health Services, NIH Bethesda, Md., Publication No. 91), as applied    to V_(HH) domains from Camelids in the article of Riechmann and    Muyldermans, J. Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195    (see for example FIG. 2 of this publication), and accordingly FR1 of    an immunoglobulin single variable domain comprises the amino acid    residues at positions 1-30, CDR1 of an immunoglobulin single    variable domain comprises the amino acid residues at positions    31-35, FR2 of an immunoglobulin single variable domain comprises the    amino acids at positions 36-49, CDR2 of an immunoglobulin single    variable domain comprises the amino acid residues at positions    50-65, FR3 of an immunoglobulin single variable domain comprises the    amino acid residues at positions 66-94, CDR3 of an immunoglobulin    single variable domain comprises the amino acid residues at    positions 95-102, and FR4 of an immunoglobulin single variable    domain comprises the amino acid residues at positions 103-113.-   v) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

1. Polypeptides of the Invention and Uses Thereof

1.1. Anti-CXCR7 Building Blocks

The polypeptides of the present invention can generally be used tomodulate, and in particular inhibit and/or prevent, binding of CXCR7 andin particular human CXCR7 (SEQ ID NO: 1) to CXCL12 (and/or CXCL11) andin particular human CXCL12 (NM_(—)000609) and/or in particular humanCXCL11 (U66096), and thus to modulate, and in particular inhibit orprevent, the signalling that is mediated by CXCR7 and in particularhuman CXCR7 (SEQ ID NO: 1) and/or CXCL12 (and/or CXCL11) and inparticular human CXCL12 (NM_(—)000609) and/or in particular human CXCL11(U66096), to modulate the biological pathways in which CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1) and/or CXCL12 (and/or CXCL11) andin particular human CXCL12 (NM_(—)000609) and/or in particular humanCXCL11 (U66096) are involved, and/or to modulate the biologicalmechanisms, responses and effects associated with such signalling orthese pathways.

As such, the polypeptides and compositions of the present invention canbe used for the diagnosis, prevention and treatment of diseases anddisorders of the present invention (herein also “diseases and disordersof the present invention”) and include, but are not limited to cancer,e.g., carcinomas, gliomas, mesotheliomas, melanomas, lymphomas,leukemias, adenocarcinomas, breast cancer, ovarian cancer, cervicalcancer, glioblastoma, leukemia, lymphoma, prostate cancer, and Burkitt'slymphoma, head and neck cancer, colon cancer, colorectal cancer,non-small cell lung cancer, small cell lung cancer, cancer of theesophagus, stomach cancer, pancreatic cancer, hepatobiliary cancer,cancer of the gallbladder, cancer of the small intestine, rectal cancer,kidney cancer, bladder cancer, prostate cancer, penile cancer, urethralcancer, testicular cancer, cervical cancer, vaginal cancer, uterinecancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenalcancer, pancreatic endocrine cancer, carcinoid cancer, bone cancer, skincancer, retinoblastomas, Hodgkin's lymphoma, non-Hodgkin's lymphoma,Kaposi's sarcoma, multicentric Castleman's disease or AIDS-associatedprimary effusion lymphoma, neuroectodermal tumors, rhabdomyosarcoma(see, Cancer, Prinicples and practice (DeVita, V. T. et al. eds 1997)for additional cancers); as well as brain and neuronal dysfunction, suchas Alzheimer's disease and multiple sclerosis; kidney dysfunction, renalallograft rejection; nasal polyposis; rheumatoid arthritis; cardiacallograft rejection; cardiac dysfunction; atherosclerosis; asthma;glomerulonephritis; contact dermatitis; inflammatory bowel disease;colitis; psoriasis; reperfusion injury; as well as other disorders anddiseases described herein. In particular, the polypeptides andcompositions of the present invention can be used for the diagnosis,prevention and treatment of diseases involving CXCR7 mediatedmetastasis, chemotaxis, cell adhesion, trans endothelial migration, cellproliferation and/or survival.

Generally, said “diseases and disorders of the present invention” can bedefined as diseases and disorders that can be diagnosed, preventedand/or treated, respectively, by suitably administering to a subject inneed thereof (i.e. having the disease or disorder or at least onesymptom thereof and/or at risk of attracting or developing the diseaseor disorder) of either a polypeptide or composition of the invention(and in particular, of a pharmaceutically active amount thereof) and/orof a known active principle active against CXCR7 and in particular humanCXCR7 (SEQ ID NO: 1) or a biological pathway or mechanism in which CXCR7and in particular human CXCR7 (SEQ ID NO: 1) is involved (and inparticular, of a pharmaceutically active amount thereof).

In particular, the polypeptides of the present invention can be used forthe diagnosis, prevention and treatment of diseases and disorders of thepresent invention which are characterized by excessive and/or unwantedCXCL12 and in particular human CXCL12 signalling mediated by CXCR7 andin particular human CXCR7 (SEQ ID NO: 1) or by the pathway(s) in whichCXCR7 and in particular human CXCR7 (SEQ ID NO: 1) is involved (e.g.CXCL11/1-TAC-CXCR7 axis). Examples of such diseases and disorders of thepresent invention will again be clear to the skilled person based on thedisclosure herein.

Thus, without being limited thereto, the immunoglobulin single variabledomains and polypeptides of the invention can for example be used todiagnose, prevent and/or to treat all diseases and disorders that arecurrently being diagnosed, prevented or treated with active principlesthat can modulate CXCR7 and in particular human CXCR7 (SEQ ID NO:1)-mediated signalling, such as those mentioned in the prior art citedabove. It is also envisaged that the polypeptides of the invention canbe used to diagnose, prevent and/or to treat all diseases and disordersfor which treatment with such active principles is currently beingdeveloped, has been proposed, or will be proposed or developed infuture. In addition, it is envisaged that, because of their favourableproperties as further described herein, the polypeptides of the presentinvention may be used for the diagnosis, prevention and treatment ofother diseases and disorders than those for which these known activeprinciples are being used or will be proposed or developed; and/or thatthe polypeptides of the present invention may provide new methods andregimens for treating the diseases and disorders described herein.

Other applications and uses of the immunoglobulin single variabledomains and polypeptides of the invention will become clear to theskilled person from the further disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of diseases and/ordisorders of the invention; and to provide methods for the diagnosis,prevention and/or treatment of such diseases and disorders that involvethe administration and/or use of such agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of diseases and/or disorders of theinvention and of the further diseases and disorders mentioned herein;and to provide methods for the diagnosis, prevention and/or treatment ofsuch diseases and disorders that involve the administration and/or theuse of such therapeutic proteins and compositions.

Accordingly, it is a specific object of the present invention to provideimmunoglobulin single variable domains that are directed against CXCR7,in particular against CXCR7 from a warm-blooded animal, more inparticular against CXCR7 from a mammal such as e.g. mouse, andespecially against human CXCR7 (SEQ ID NO: 1); and to provide proteinsand polypeptides comprising or essentially consisting of at least onesuch immunoglobulin single variable domain.

In particular, it is a specific object of the present invention toprovide such immunoglobulin single variable domains and such proteinsand/or polypeptides that are suitable for prophylactic, therapeuticand/or diagnostic use in a warm-blooded animal, and in particular in amammal, and more in particular in a human being.

More in particular, it is a specific object of the present invention toprovide such immunoglobulin single variable domains and such proteinsand/or polypeptides that can be used for the prevention, treatment,alleviation and/or diagnosis of one or more diseases, disorders orconditions associated with CXCR7 and/or mediated by CXCR7 (such as thediseases, disorders and conditions mentioned herein) in a warm-bloodedanimal, in particular in a mammal, and more in particular in a humanbeing.

It is also a specific object of the invention to provide suchimmunoglobulin single variable domains and such proteins and/orpolypeptides that can be used in the preparation of pharmaceutical orveterinary compositions for the prevention and/or treatment of one ormore diseases, disorders or conditions associated with and/or mediatedby CXCR7 (such as the diseases, disorders and conditions mentionedherein) in a warm-blooded animal, in particular in a mammal, and more inparticular in a human being.

In the invention, generally, these objects are achieved by the use ofthe immunoglobulin single variable domains, proteins, polypeptides andcompositions that are described herein.

In general, the invention provides immunoglobulin single variabledomains that are directed against (as defined herein) and/or canspecifically bind (as defined herein) to CXCR7 and in particular humanCXCR7 (SEQ ID NO: 1); as well as compounds and constructs, and inparticular proteins and polypeptides, that comprise at least one suchamino acid sequence.

More in particular, the invention provides immunoglobulin singlevariable domains and polypeptides that can bind to CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1) with an affinity (suitablymeasured and/or expressed as a K_(D)-value (actual or apparent), aK_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate,or alternatively as an IC₅₀ value, as further described herein) that isas defined herein; as well as compounds and constructs, and inparticular proteins and polypeptides, that comprise at least one suchamino acid sequence.

In particular aspect, the immunoglobulin single variable domains and/orpolypeptides of the invention are such that they:

-   -   bind to human CXCR7 (SEQ ID NO:1) with an EC50 of 100 nM or        lower, more preferably of 50 nM or lower, even more preferably        of 20 nM or lower, most preferably of 10 nM or lower in a        binding FACS assay as e.g. described in the experimental part        (see example 8), and wherein the polypeptides comprise only one        human CXCR7 binding immunoglobulin single variable domain unit;        and/or such that they:    -   fully displace human CXCL12 (SDF-1) from human CXCR7 (SEQ ID        NO: 1) at an average Ki value of 100 nM or less, more preferably        at an average Ki value of 20 nM or less, even more preferably at        an average Ki value of 10 nM or less in an assay as e.g.        described in the experimental part (examples 9 and 10), and        wherein the polypeptides comprise only one human CXCR7 binding        immunoglobulin single variable domain unit, and wherein full        displacement means an average CXCL12 displacement of about 60%        to 80% and more (e.g. when measured according to the ligand        displacement assay of example 9) or wherein full displacement        means an average CXCL12 displacement of about 80% to 100% and        more (when measured according to the FACS based competition        assay of example 10);        and/or such that they:    -   fully displace human CXCL11 (I-TAC) from human CXCR7 (SEQ ID        NO: 1) at an average Ki value of 1000 nM or less, more        preferably at an average Ki value 500 nM or less, even more        preferably at an average Ki value 100 nM or less, even more        preferably at an average Ki value of 20 nM or less, even more        preferably at an average Ki value of 10 nM or less in an assay        as e.g. described in the experimental part (examples 9 and 10),        and wherein the polypeptides comprise only one human CXCR7        binding immunoglobulin single variable domain unit, and wherein        full displacement means an average CXCL11 displacement of about        60% to 80% and more (e.g. when measured according to the ligand        displacement assay of example 9) or wherein full displacement        means an average CXCL12 displacement of about 80% to 100% and        more (when measured according to the FACS based competition        assay of example 10) and/or such that they:    -   partially displace human CXCL12 (SDF-1) from human CXCR7 (SEQ ID        NO: 1) at an average Ki value of 100 nM or less, more preferably        at an average Ki value of 20 nM or less, even more preferably at        an average Ki value of 10 nM or less in an assay as e.g.        described in the experimental part (examples 9 and 10), and        wherein the polypeptides comprise only one human CXCR7 binding        immunoglobulin single variable domain unit, and wherein partial        displacement means an average CXCL12 displacement of about 40%        to 60% (e.g. when measured according to the ligand displacement        assay of example 9) or wherein partial displacement means an        average CXCL12 displacement of about 50% to 80% (when measured        according to the FACS based competition assay of example 10);        and/or such that they:

partially displace human CXCL11 (I-TAC) from human CXCR7 (SEQ ID NO: 1)at an average Ki value of 1000 nM or less, more preferably at an averageKi value 500 nM or less, even more preferably at an average Ki value 100nM or less, even more preferably at an average Ki value of 20 nM orless, even more preferably at an average Ki value of 10 nM or less in anassay as e.g. described in the experimental part (examples 9 and 10),and wherein the polypeptides comprise only one human CXCR7 bindingimmunoglobulin single variable domain unit, and wherein partialdisplacement means an average CXCL11 displacement of about 40% to 60%(e.g. when measured according to the ligand displacement assay ofexample 9) or wherein partial displacement means an average CXCL12displacement of about 50% to 80% (when measured according to the FACSbased competition assay of example 10). Some preferred technical valuesfor binding, displacing, migration or other in vivo and/or in vitropotency of the immunoglobulin single variable domains or polypeptides ofthe invention to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) willbecome clear from the further description and examples herein.

For binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), anamino acid sequence of the invention will usually contain within itsamino acid sequence one or more amino acid residues or one or morestretches of amino acid residues (i.e. with each “stretch” comprisingtwo or amino acid residues that are adjacent to each other or in closeproximity to each other, i.e. in the primary or tertiary structure ofthe amino acid sequence) via which the amino acid sequence of theinvention can bind to CXCR7 and in particular human CXCR7 (SEQ ID NO:1), which amino acid residues or stretches of amino acid residues thusform the “site” for binding to CXCR7 and in particular human CXCR7 (SEQID NO: 1) (also referred to herein as the “antigen binding site”).

The immunoglobulin single variable domains provided by the invention arepreferably in essentially isolated form (as defined herein), or formpart of a protein or polypeptide of the invention (as defined herein),which may comprise or essentially consist of one or more immunoglobulinsingle variable domains of the invention and which may optionallyfurther comprise one or more further immunoglobulin single variabledomains (all optionally linked via one or more suitable linkers), and/orone or more further binding domains, binding units, amino acid sequencesor other (functional) groups or moieties, that preferably also conferone or more desired properties to the constructs (some non-limitingexamples of the same will become clear from the further descriptionherein). Again, such further binding domains, binding units, amino acidsequences or other (functional) groups or moieties may for example beone or more other immunoglobulin single variable domains, such as one ormore (single) domain antibodies, dAb's or Nanobodies (e.g. a V_(HH),humanized V_(HH) or camelized V_(H), such as a camelised human V_(H)),so as to provide a “bispecific” protein or polypeptide of the invention(i.e. a polypeptide of the invention that contains at least one—such asone or two—immunoglobulin single variable domain that is directedagainst CXCR7 and at least one—such as one or two—immunoglobulin singlevariable domain that is directed against another target).

For example, according to a specific but non-limiting aspect, theconstructs, proteins or polypeptides of the invention may have beenprovided with an increased half-life, for example by functionalisationand/or by including in the construct a moiety or binding unit thatincreases the half-life of the construct. Examples of suchfunctionalisation, moieties or binding units will be clear to theskilled person and may for example include pegylation, fusion to serumalbumin, or fusion to a peptide or binding unit that can bind to a serumprotein such as serum albumin

In the latter constructs (i.e. fusion constructs comprising at leastone—such as one or two—amino acid sequence of the invention and at leastone—such as one or two—peptide or binding unit that can bind to a serumprotein such as serum albumin), the serum-albumin binding peptide orbinding domain may be any suitable serum-albumin binding peptide orbinding domain capable of increasing the half-life of the construct(compared to the same construct without the serum-albumin bindingpeptide or binding domain), and may in particular be serum albuminbinding peptides as described in WO 2008/068280 by applicant (and inparticular WO 2009/127691 and the non-prepublished U.S. application61/301,819, both by applicant), or a serum-albumin bindingimmunoglobulin single variable domain (such as a serum-albumin bindingNanobody; for example Alb-1 or a humanized version of Alb-1 such asAlb-8, for which reference is for example made to WO 06/122787).

With respect to half-life, it should be noted that in the invention, andby using the various half-life extending techniques described herein(for example, by suitably choosing a serum-albumin binding peptideaccording to WO 2008/068280, WO 2009/127691 and/or the non-prepublishedU.S. application 61/301,819), the half-life of a construct orpolypeptide of the invention can (and preferably is) suitably “tailored”for the intended (therapeutic and/or diagnostic) application and/or toobtain the best balance between the desired therapeutic and/orpharmacological effect and possible undesired side-effects.

Thus, for example, and without limitation, a preferred aspect of theinvention provides a “bispecific” polypeptide consisting essentially ofone immunoglobulin single variable domain directed against human CXCR7(or, alternatively, of two immunoglobulin single variable domainsdirected against human CXCR7, which may be the same or different, i.e.so as to provide—when they are the same or different—a “bivalent”polypeptide of the invention, or—when they are different—“biparatopic”polypeptide of the invention) and one immunoglobulin single variabledomain directed against human serum albumin linked by a peptide linker(as defined herein), so as to provide a bispecific polypeptide of theinvention, respectively, all as described herein. Such a protein orpolypeptide may also be in essentially isolated form (as definedherein).

In another specific, but non-limiting aspect, an amino acid sequence(such as a Nanobody) of the invention or a polypeptide of the invention(such as a bivalent, biparatopic or bispecific polypeptide of theinvention) may be suitably linked (again, chemically or via one or moresuitable linkers or spacers) to a toxin or to a (cyto)toxic residue,moiety or payload. Examples of suitable (cyto)toxic moieties, compounds,payloads or residues which can be linked to amino acids sequences orpolypeptides of the invention to provide—for example—a cytotoxiccompound (i.e. an antibody-drug conjugate or “ADC” based upon an aminoacid sequence or polypeptide of the invention) will be clear to theskilled person. Reference is for example made to the review by Ducry andStump, Bioconjugate Chem., 2010, 21 (1), pp 5-13. Such cytotoxic aminoacid sequences or polypeptides of the invention may in particular beuseful/suitable for those applications in which it is intended to kill acell that expresses the target against which the amino acid sequences orpolypeptides of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell. Usually, but without limitation, (cyto)toxic polypeptides of theinvention will either not be half-life extended or will have only alimited and/or tightly controlled half-life extension.

In another aspect, at least one amino acid sequence of the invention(i.e. immunoglobulin single variable domain against CXCR7) may besuitably linked to at least one immunoglobulin single variable domainthat is directed against CXCR4, so as to provide a bispecificpolypeptide of the invention that is directed against both CXCR7 andCXCR4.

For example, in this aspect, at least one—such as one or two—amino acidsequences of the invention may be suitably linked to at least one—suchas one or two—immunoglobulin single variable domains against CXCR4.

Some preferred but non-limiting examples of immunoglobulin singlevariable domains against CXCR4 that can be used in such constructs are(or may be suitably chosen from)

-   -   the immunoglobulin single variable domains (and in particular        one of the Nanobodies) against CXCR4 from the international        application WO 09/138519 by Ablynx N.V. (for example and without        limitation, 238D2/SEQ ID NO: 238 and 238D4/SEQ ID NO: 239 in        Table B-1.1 of WO 09/138519); and/or    -   the sequence-optimized/improved variants of the amino acid        sequences 238D2 and 238D4 described in the non-prepublished U.S.        application 61/358,495 by Ablynx N.V. filed on Jun. 25, 2010;        and/or    -   the immunoglobulin single variable domains that are capable of        binding to the same epitope as 238D2 and/or 238D4 as described        in the PCT application PCT/EP210/064766 by Ablynx N.V. filed on        Oct. 4, 2010; and/or    -   the 10E9-type sequences, 281E10-type sequences, 10E12-type        sequences, 10A10-type sequences, 10G10-type sequences, 14A2-type        sequences, 15A1-type sequences, 15H3-type sequences and/or        283B6-type sequences described on pages 7-13 of the PCT        application PCT/EP2011/050156 by Ablynx N.V. filed on Jan. 7,        2011; and/or    -   the 10E9-type sequences, 281E10-type sequences, 10E12-type        sequences, 10A10-type sequences, 10G10-type sequences, 14A2-type        sequences, 15A1-type sequences, 15H3-type sequences and/or        283B6-type sequences described on pages 15-47 of the PCT        application PCT/EP2011/050156 by Ablynx N.V. filed on Jan. 7,        2011.

The above anti-CXCR7/CXCR4 bispecific constructs (as well as otherbispecific constructs comprising at least one amino acid sequence of theinvention) may again be suitably half-life extended (i.e. by pegylation,fusion to serum albumin, or fusion to a peptide or binding unit that canbind to a serum protein such as serum albumin, as further describedherein), and thus may for example further comprise a serum-albuminbinding peptide or binding domain (such as those described herein),optionally linked via one or more suitable spacers or linkers.

Thus, one specific but non-limiting aspect of the invention is apolypeptide that comprises one or two (and preferably one)immunoglobulin single variable domains (as defined herein, andpreferably one or two Nanobodies) against CXCR7, one or two (andpreferably one) immunoglobulin single variable domains (as definedherein, and preferably one or two Nanobodies) against CXCR4, and apeptide or immunoglobulin single variable domain against (human) serumalbumin, optionally suitably linked via one or more spacers or linkers.

The above anti-CXCR7/CXCR4 bispecific constructs (as well as otherbispecific constructs comprising at least one amino acid sequence of theinvention) may also be suitably linked (again, chemically or via one ormore suitable linkers or spacers) to a toxin or to a (cyto)toxicresidue, moiety or payload (as further described herein). Again, such(cyto)toxic bispecfic polypeptides of the invention will either not behalf-life extended or will have only a limited and/or tightly controlledhalf-life extension.

The invention in its broadest sense also comprises derivatives of theamino acid sequences (e.g., Nanobodies) of the invention and of thepolypeptides of the invention. Such derivatives can generally beobtained by modification, and in particular by chemical and/orbiological (e.g., enzymatical) modification, of the amino acid sequences(e.g. Nanobodies) of the invention and polypeptides of the inventionand/or of one or more of the amino acid residues that form theNanobodies of the invention.

Examples of such modifications, as well as examples of amino acidresidues within the amino acid sequences (e.g. Nanobodies) of theinvention and polypeptides that can be modified in such a manner (i.e.either on the protein backbone but preferably on a side chain), methodsand techniques that can be used to introduce such modifications and thepotential uses and advantages of such modifications will be clear to theskilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the amino acidsequences (e.g. Nanobodies) of the invention and polypeptides of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to, thefluorescent labels, phosphorescent labels, chemiluminescent labels,bioluminescent labels, radio-isotopes, metals, metal chelates, metalliccations, chromophores and enzymes, such as those mentioned on page 109of WO 08/020079. Other suitable labels will be clear to the skilledperson, and for example include moieties that can be detected using NMRor ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

The immunoglobulin single variable domains and polypeptides of theinvention as such preferably essentially consist of a single amino acidchain that is not linked via disulphide bridges to any other amino acidsequence or chain (but that may or may not contain one or moreintramolecular disulphide bridges. For example, it is known that agentof the invention—as described herein—may sometimes contain a disulphidebridge between CDR3 and CDR1 or FR2). However, it should be noted thatone or more immunoglobulin single variable domains of the invention maybe linked to each other and/or to other immunoglobulin single variabledomains (e.g. via disulphide bridges) to provide peptide constructs thatmay also be useful in the invention (for example Fab′ fragments, F(ab′)₂fragments, ScFv constructs, “diabodies” and other multispecificconstructs. Reference is for example made to the review by Holliger andHudson, Nat. Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, is in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the immunoglobulin single variable domains of the invention (as well ascompounds, constructs and polypeptides comprising the same) arepreferably directed against human CXCR7 and in particular human CXCR7(SEQ ID NO: 1); whereas for veterinary purposes, the immunoglobulinsingle variable domains and polypeptides of the invention are preferablydirected against CXCR7 from the species to be treated, or at leastcross-reactive with CXCR7 from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against CXCR7and in particular human CXCR7 (SEQ ID NO: 1), contain one or morefurther binding sites for binding against other antigens, proteins ortargets.

The efficacy of the immunoglobulin single variable domains andpolypeptides of the invention, and of compositions comprising the same,can be tested using any suitable in vitro assay, cell-based assay, invivo assay and/or animal model known per se, or any combination thereof,depending on the specific disease or disorder involved. Suitable assaysand animal models will be clear to the skilled person, and for exampleinclude ligand displacement assays (Burns et al, J Exp Med. 2006 4;203(9):2201-13), beta arrestin recruitment assays (Zabel et al JImmunol. 2009 1; 183(5):3204-11), dimerization assays (Luker et al,FASEB J 2009; 23(3):823-34), signaling assays (Wang et al, J. Immunol.2009 Sep. 1; 183(5):3204-11) proliferation assays (Wang et al, J.Immunol. 2009 Sep. 1; 183(5):3204-11; Odemis et al., J Cell Sign. 2010Apr. 1; 123(Pt 7): 1081-8), survival assays (Burns et al, J Exp Med.2006 4; 203(9):2201-13), cell adhesion assays (Burns et al, J Exp Med.2006 4; 203(9):2201-13) and transendothelial migration assays (Mazzinghiet al, J Exp Med. 2008 Feb. 18; 205(2):479-90), endothelial cellsprouting assays (Wang et al, J. Immunol. 2009 Sep. 1; 183(5):3204-11),myogenic differentiation (Melchionna et al., Muscle Nerve, 2010 Feb. 11)and in vivo xenograft models (Burns et al, J Exp Med. 2006 4;203(9):2201-13), collagen induced arthritis models (Hegen et al, AnnRheum Dis. 2008 November; 67(11):1505-15) and experimental autoimmuneencephalomyelitis models (Wekerle, Ann Rheum Dis. 2008 December; 67Suppl 3:iii56-60) as well as the assays and animal models used in theexperimental part below and in the prior art cited herein.

Also, according to the invention, immunoglobulin single variable domainsand polypeptides that are directed against CXCR7 from a first species ofwarm-blooded animal may or may not show cross-reactivity with CXCR7 fromone or more other species of warm-blooded animal. For example,immunoglobulin single variable domains and polypeptides directed againsthuman CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) may or may notshow cross reactivity with CXCR7 from one or more other species ofprimates (such as, without limitation, monkeys from the genus Macaca(such as, and in particular, cynomolgus monkeys (Macaca fascicularis)and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus))and/or with CXCR7 from one or more species of animals that are oftenused in animal models for diseases (for example mouse, rat, rabbit, pigor dog), and in particular in animal models for diseases and disordersassociated with CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) (suchas the species and animal models mentioned herein). In this respect, itwill be clear to the skilled person that such cross-reactivity, whenpresent, may have advantages from a drug development point of view,since it allows the immunoglobulin single variable domains andpolypeptides against human CXCR7 and in particular human CXCR7 (SEQ IDNO: 1) to be tested in such disease models.

More generally, immunoglobulin single variable domains and polypeptidesof the invention that are cross-reactive with CXCR7 from multiplespecies of mammal will usually be advantageous for use in veterinaryapplications, since it will allow the same amino acid sequence orpolypeptide to be used across multiple species. Thus, it is alsoencompassed within the scope of the invention that immunoglobulin singlevariable domains and polypeptides directed against CXCR7 from onespecies of animal (such as immunoglobulin single variable domains andpolypeptides against human CXCR7 (SEQ ID NO: 1)) can be used in thetreatment of another species of animal, as long as the use of theimmunoglobulin single variable domains and/or polypeptides provide thedesired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1) against which the immunoglobulinsingle variable domains and polypeptides of the invention are directed.For example, the immunoglobulin single variable domains and polypeptidesmay or may not be directed against the CXCL11/CXCL12 interaction siteand/or CXCR7/CXCR7 homodimerization site and/or CXCR4/CXCR7heterodimerization site (or heterodimerization of CXCR7 to otherchemokine receptor such as e.g. CXCR3), and are as further definedherein.

As further described herein, a polypeptide of the invention may contain(although not preferred) two or more immunoglobulin single variabledomains of the invention that are directed against CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1). Generally, such polypeptides willbind to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) withincreased avidity compared to a single amino acid sequence of theinvention. Such a polypeptide may for example comprise twoimmunoglobulin single variable domains of the invention that aredirected against the same antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of CXCR7 and in particularhuman CXCR7 (SEQ ID NO: 1) (which may or may not be an interactionsite); or comprise at least one “first” amino acid sequence of theinvention that is directed against a first same antigenic determinant,epitope, part, domain, subunit or confirmation (where applicable) ofCXCR7 and in particular human CXCR7 (SEQ ID NO: 1) (which may or may notbe an interaction site); and at least one “second” amino acid sequenceof the invention that is directed against a second antigenicdeterminant, epitope, part, domain, subunit or confirmation (whereapplicable) different from the first (and which again may or may not bean interaction site). Preferably, in such “biparatopic” polypeptides ofthe invention, at least one amino acid sequence of the invention isdirected against an interaction site (as defined herein), although theinvention in its broadest sense is not limited thereto. E.g.polypeptides of the invention may be formatted e.g. in a biparatopic waysuch as to combine monovalent building blocks directed against differentepitopes as characterized in the experimental part (see examples 9 to12).

Also, when the target is part of a binding pair (for example, areceptor-ligand binding pair), the immunoglobulin single variabledomains and polypeptides may be such that they compete with the cognatebinding partners, e.g. CXCL11 (also referred to as I-TAC) and/or CXCL12(also referred to as SDF-1), for binding to CXCR7, and/or such that they(fully or partially) neutralize binding of the binding partner to thetarget.

It is also expected that the immunoglobulin single variable domains andpolypeptides of the invention will generally bind to all naturallyoccurring or synthetic analogs, variants, mutants, alleles, parts andfragments of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1); or atleast to those analogs, variants, mutants, alleles, parts and fragmentsof CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) that contain oneor more antigenic determinants or epitopes that are essentially the sameas the antigenic determinant(s) or epitope(s) to which theimmunoglobulin single variable domains and polypeptides of the inventionbind to CXCR7 and in particular to human CXCR7 (SEQ ID NO: 1). Again, insuch a case, the immunoglobulin single variable domains and polypeptidesof the invention may bind to such analogs, variants, mutants, alleles,parts and fragments with an affinity and/or specificity that are thesame as, or that are different from (i.e. higher than or lower than),the affinity and specificity with which the immunoglobulin singlevariable domains of the invention bind to (wild-type) CXCR7.

As CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) exists in amonomeric form and in one or more multimeric forms, e.g. in homodimericas well in heterodimeric form with CXCR4, e.g. human CXCR4 (R M Maksymet al., supra; K E Luker et al. supra), it is within the scope of theinvention that the immunoglobulin single variable domains andpolypeptides of the invention i) only bind to CXCR7 and in particularhuman CXCR7 (SEQ ID NO: 1) in monomeric form, ii) only bind to CXCR7 andin particular human CXCR7 (SEQ ID NO: 1) in multimeric/dimeric (homo-and/or heterodimeric) form, or iii) bind to both the monomeric and themultimeric form. In a preferred aspect of the invention, thepolypeptides of the invention prevent formation of homodimeric humanCXCR7 complexes and/or heterodimeric human CXCR4/CXCR7 complexes. Inanother preferred aspect of the invention, the polypeptides of theinvention do not induce (even at higher concentration such as 10 nM orless, 50 nM or less, 100 nM or less, or 500 nM or less) formation ofhomodimeric human CXCR7 complexes and/or heterodimeric human CXCR4/CXCR7complexes. Again, in such a case, the polypeptides of the invention maybind to the monomeric form with an affinity and/or specificity that arethe same as, or that are different from (i.e. higher than or lowerthan), the affinity and specificity with which the immunoglobulin singlevariable domains of the invention bind to the multimeric form.

Also, when CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) canassociate with other proteins or polypeptides to form protein complexes(e.g. with CXCL12/SDF-1 or CXCL11/1-TAC), it is within the scope of theinvention that the immunoglobulin single variable domains andpolypeptides of the invention bind to CXCR7 and in particular humanCXCR7 (SEQ ID NO: 1) in its non-associated state (and e.g. prevent theligand binding), bind to CXCR7 and in particular human CXCR7 (SEQ IDNO: 1) in its associated state, or bind to both (preferably to thenon-associated state). In all these cases, the immunoglobulin singlevariable domains and polypeptides of the invention may bind to suchassociated protein complexes with an affinity and/or specificity thatmay be the same as or different from (i.e. higher than or lower than)the affinity and/or specificity with which the immunoglobulin singlevariable domains and polypeptides of the invention bind to CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1) in its non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more immunoglobulin single variable domains directedagainst CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) may bind withhigher avidity to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1)than the corresponding monomeric amino acid sequence(s). For example,and without limitation, proteins or polypeptides that contain two ormore immunoglobulin single variable domains directed against differentepitopes of CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) may (andusually will) bind with higher avidity than each of the differentmonomers, and proteins or polypeptides that contain two or moreimmunoglobulin single variable domains directed against CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1) may (and usually will) bind alsowith higher avidity to a multimer (e.g. homodimer, heterodimer withCXCR4) of CXCR7 and in particular to a multimer (e.g. homodimer,heterodimer with human CXCR4) of human CXCR7 (SEQ ID NO: 1).

Generally, immunoglobulin single variable domains and polypeptides ofthe invention will at least bind to those forms of CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1) (including monomeric, multimeric,associated and different conformational forms) that are the mostrelevant from a biological and/or therapeutic point of view, as will beclear to the skilled person.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of theimmunoglobulin single variable domains and polypeptides of theinvention, and/or to use proteins or polypeptides comprising oressentially consisting of one or more of such parts, fragments, analogs,mutants, variants, alleles and/or derivatives, as long as these aresuitable for the uses envisaged herein. Such parts, fragments, analogs,mutants, variants, alleles and/or derivatives will usually contain (atleast part of) a functional antigen-binding site for binding againstCXCR7 and in particular human CXCR7 (SEQ ID NO: 1); and more preferablywill be capable of specific binding to CXCR7 and in particular humanCXCR7 (SEQ ID NO: 1), and even more preferably capable of binding toCXCR7 and in particular human CXCR7 (SEQ ID NO: 1) with an EC50 value,average Ki, IC₅₀ value concerning binding, migration, displacing and/orproliferation blocking and/or other measures for potency, as furtherdescribed herein, e.g. in the experimental part) that is as definedherein and such parts, fragments, analogs, mutants, variants, allelesand/or derivatives may be more potent, more stable, more soluble and mayhave the same epitope. Some non-limiting examples of such parts,fragments, analogs, mutants, variants, alleles, derivatives, proteinsand/or polypeptides will become clear from the further descriptionherein. Additional fragments or polypeptides of the invention may alsobe provided by suitably combining (i.e. by linking or genetic fusion)one or more (smaller) parts or fragments as described herein.

For a general description of immunoglobulin single variable domains,reference is made to the further description below, as well as to theprior art cited herein. In this respect, it should however be noted thatthis description and the prior art mainly describes immunoglobulinsingle variable domains of the so-called “V_(H)3 class” (i.e.immunoglobulin single variable domains with a high degree of sequencehomology to human germline sequences of the V_(H)3 class such as DP-47,DP-51 or DP-29), which form a preferred aspect of this invention. Itshould however be noted that the invention in its broadest sensegenerally covers any type of immunoglobulin single variable domainsdirected against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1), andfor example also covers the immunoglobulin single variable domainsbelonging to the so-called “V_(H)4 class” (i.e. immunoglobulin singlevariable domains with a high degree of sequence homology to humangermline sequences of the V_(H)4 class such as DP-78), as for exampledescribed in WO 07/118670.

Generally, immunoglobulin single variable domains (in particular V_(HH)sequences and sequence optimized immunoglobulin single variable domains)can in particular be characterized by the presence of one or more“Hallmark residues” (as described herein) in one or more of theframework sequences (again as further described herein).

Thus, generally, an immunoglobulin single variable domain can be definedas an amino acid sequence with the (general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively.

In a preferred aspect, the invention provides polypeptides comprising atleast an immunoglobulin single variable domain that is an amino acidsequence with the (general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) at least one of the amino acid residues at positions 11, 37, 44,    45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering    are chosen from the Hallmark residues mentioned in Table A-1 below;    and in which:-   ii) said amino acid sequence has at least 80%, more preferably 90%,    even more preferably 95% amino acid identity with at least one of    the immunoglobulin single variable domains as shown in WO    2009/138519 (see SEQ ID NO's: 1 to 125 in WO 2009/138519), in which    for the purposes of determining the degree of amino acid identity,    the amino acid residues that form the CDR sequences (indicated with    X in the sequences) are disregarded; and in which:-   iii) the CDR sequences are generally as further defined herein (e.g.    the CDR1, CDR2 and CDR3 in a combination as provided in Table (B-2),    note that the CDR definitions are calculated according to the Kabat    numbering system).

TABLE A-1 Hallmark Residues in VHHs Position Human V_(H)3 HallmarkResidues  11 L, V; L, S, V, M, W, F, T, Q, E, A, R, predominantly L G,K, Y, N, P, I; preferably L  37 V, I, F; usually V F⁽¹⁾, Y, V, L, A, H,S, I, W, C, N, G, D, T, P, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G E⁽³⁾, Q⁽³⁾,G⁽²⁾, D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾, E⁽³⁾ orQ⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H, F, G, Q,S, E, T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, Y F⁽¹⁾, L⁽¹⁾or W⁽²⁾ G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferablyW⁽²⁾, L⁽¹⁾ or F⁽¹⁾  83 R or K; usually R R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I,V, G, M, L, A, D, Y, H; preferably K or R; most preferably K  84 A, T,D; P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, predominantly A Y, N, Q, G, E;preferably P 103 W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, Q, P⁽⁶⁾,E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108L, M or T; Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H; predominantly Lpreferably Q or L⁽⁷⁾ ⁽¹⁾In particular, but not exclusively incombination with KERE or KQRE at positions 43-46. ⁽²⁾Usually as GLEW atpositions 44-47. ⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. asKEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47.Alternatively, also sequences such as TERE (for example TEREL), TQRE(for example TQREL), KECE (for example KECEL or KECER), KQCE (forexample KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREFor RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL orQQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.Some other possible, but less preferred sequences include for exampleDECKL and NVCEL. ⁽⁴⁾With both GLEW at positions 44-47 and KERE or KQREat positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturallyoccurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, incombination with GLEW at positions 44-47. ⁽⁷⁾With the proviso that whenpositions 44-47 are GLEW, position 108 is always Q in (non-humanized)V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group alsocontains GLEW-like sequences at positions 44-47, such as for exampleGVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER andELEW

Again, such immunoglobulin single variable domains may be derived in anysuitable manner and from any suitable source, and may for example benaturally occurring V_(HH) sequences (i.e. from a suitable species ofCamelid, e.g. llama) or synthetic or semi-synthetic VHs or VLs (e.g.from human). Such immunoglobulin single variable domains may include“humanized” or otherwise “sequence optimized” VHHs, “camelized”immunoglobulin sequences (and in particular camelized heavy chainvariable domain sequences, i.e. camelized VHs), as well as human VHs,human VLs, camelid VHHs that have been altered by techniques such asaffinity maturation (for example, starting from synthetic, random ornaturally occurring immunoglobulin sequences), CDR grafting, veneering,combining fragments derived from different immunoglobulin sequences, PCRassembly using overlapping primers, and similar techniques forengineering immunoglobulin sequences well known to the skilled person;or any suitable combination of any of the foregoing as further describedherein.

In a further preferred aspect, the invention provides polypeptidescomprising one immunoglobulin single variable domain with amino acidsequence selected from the group consisting of amino acid sequences withSEQ ID NO's: 39 to 43 (see Table B-2) and one immunoglobulin singlevariable domain with amino acid sequence selected from the groupconsisting of moieties providing an increased half-life (see below).

In a further preferred aspect, the invention provides polypeptidescomprising at least an immunoglobulin single variable domain with aminoacid sequence selected from the group consisting of amino acid sequencesthat essentially consist of 4 framework regions (FR1 to FR4,respectively) and 3 complementarity determining regions (CDR1 to CDR3,respectively), in which the CDR sequences of said amino acid sequenceshave at least 70% amino acid identity, preferably at least 80% aminoacid identity, more preferably at least 90% amino acid identity, such as95% amino acid identity or more or even essentially 100% amino acididentity with the CDR sequences of at least one of the immunoglobulinsingle variable domains of SEQ ID NO's: 39 to 43 (see Tables B-2 andB-3). This degree of amino acid identity can for example be determinedby determining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 39 to 43 (see Tables B-2 and B-3), in whichthe amino acid residues that form the framework regions are disregarded.Such polypeptides and/or immunoglobulin single variable domains of theinvention may further provide the following:

-   1. Polypeptides comprising at least one (preferably one)    immunoglobulin single variable domain that is directed against (as    defined herein) CXCR7 and in particular human CXCR7 (SEQ ID NO: 1)    and that has at least 80%, preferably at least 85%, such as 90% or    95% or more sequence identity with at least one of the    immunoglobulin single variable domains of SEQ ID NO's: 39 to 43 (see    Table B-3);-   2. Polypeptides comprising at least one (preferably one)    immunoglobulin single variable domain that is directed against (as    defined herein) CXCR7 and in particular human CXCR7 (SEQ ID NO: 1)    and that cross-block (as defined herein) the binding of at least one    of the immunoglobulin single variable domains of SEQ ID NO's: 39 to    43 (see Table B-3) to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) and/or that compete with at least one of the immunoglobulin    single variable domains of SEQ ID NO's: 39 to 43 (see Table B-3) for    binding to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1); and-   3. which immunoglobulin single variable domains may be as further    described herein; as well as polypeptides of the invention that    comprise one or more (preferably one) of such immunoglobulin single    variable domains (which may be as further described herein, and may    for example be bispecific (e.g. also bind to serum albumin) and/or    biparatopic polypeptides as described herein), and nucleic acid    sequences that encode such immunoglobulin single variable domains    and polypeptides. Such immunoglobulin single variable domains and    polypeptides do not include any naturally occurring ligands.

The polypeptides of the invention comprise or essentially consist of atleast one immunoglobulin single variable domain of the invention. Somepreferred, but non-limiting examples of immunoglobulin single variabledomains of the invention are given in SEQ ID NO's: 39 to 43 (see TableB-3).

1.2. Serum Albumin Binding Building Blocks or Other Building BlocksIncreasing Half-Life

In another aspect, the invention relates to a compound or construct, andin particular to a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or more(preferably one) immunoglobulin single variable domains directed tohuman CXCR7 (or suitable fragments thereof), and optionally furthercomprises one or more other groups, residues, moieties or binding units.As will become clear to the skilled person from the further disclosureherein, such further groups, residues, moieties, binding units orimmunoglobulin single variable domains may or may not provide furtherfunctionality to the amino acid sequence of the invention (and/or to thecompound or construct in which it is present) and may or may not modifythe properties of the amino acid sequence of the invention.

As will be clear from the further description above and herein, thismeans that the immunoglobulin single variable domains of the inventioncan be used as “building blocks” to form polypeptides of the invention,i.e. by suitably combining them with other groups, residues, moieties orbinding units, in order to form compounds or constructs as describedherein (such as, without limitations, the biparatopic, bi/multivalentand bi/multispecific polypeptides of the invention described herein)which combine within one molecule one or more desired properties orbiological functions.

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more immunoglobulin single variable domains of the invention tothe one or more further groups, residues, moieties or binding units,optionally via the one or more suitable linkers, so as to provide thecompound or polypeptide of the invention. Polypeptides of the inventioncan also be prepared by a method which generally comprises at least thesteps of providing a nucleic acid that encodes a polypeptide of theinvention, expressing said nucleic acid in a suitable manner, andrecovering the expressed polypeptide of the invention. Such methods canbe performed in a manner known per se, which will be clear to theskilled person, for example on the basis of the methods and techniquesfurther described herein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedimmunoglobulin single variable domains form a further aspect of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional immunoglobulin single variable domains,such that the compound or construct is a (fusion) protein or (fusion)polypeptide. In a preferred but non-limiting aspect, said one or moreother groups, residues, moieties or binding units are immunoglobulinsequences. Even more preferably, said one or more other groups,residues, moieties or binding units are chosen from the group consistingof domain antibodies, immunoglobulin single variable domains that aresuitable for use as a domain antibody, single domain antibodies,immunoglobulin single variable domains that are suitable for use as asingle domain antibody, “dAb”'s, immunoglobulin single variable domainsthat are suitable for use as a dAb, or Nanobodies. Alternatively, suchgroups, residues, moieties or binding units may for example be chemicalgroups, residues, moieties, which may or may not by themselves bebiologically and/or pharmacologically active. For example, and withoutlimitation, such groups may be linked to the one or more immunoglobulinsingle variable domains of the invention so as to provide a “derivative”of an amino acid sequence or polypeptide of the invention, as furtherdescribed herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are immunoglobulin singlevariable domains. In the compounds or constructs described above, theone or more immunoglobulin single variable domains of the invention andthe one or more groups, residues, moieties or binding units may belinked directly to each other and/or via one or more suitable linkers orspacers. For example, when the one or more groups, residues, moieties orbinding units are immunoglobulin single variable domains, the linkersmay also be immunoglobulin single variable domains, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

In one specific, but non-limiting aspect of the invention, which will befurther described herein, the polypeptides of the invention have anincreased half-life in serum (as further described herein) compared tothe immunoglobulin single variable domain from which they have beenderived. For example, an immunoglobulin single variable domain of theinvention may be linked (chemically or otherwise) to one or more groupsor moieties that extend the half-life (such as PEG), so as to provide aderivative of an amino acid sequence of the invention with increasedhalf-life.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise immunoglobulin single variable domainsor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);immunoglobulin single variable domains of the invention that comprise atleast one additional binding site for binding to a serum protein (suchas serum albumin); or polypeptides of the invention that comprise atleast one amino acid sequence of the invention that is linked to atleast one moiety (and in particular at least one amino acid sequence)that increases the half-life of the amino acid sequence of theinvention. Examples of polypeptides of the invention that comprise suchhalf-life extending moieties or immunoglobulin single variable domainswill become clear to the skilled person based on the further disclosureherein; and for example include, without limitation, polypeptides inwhich the one or more immunoglobulin single variable domains of theinvention are suitably linked to one or more serum proteins or fragmentsthereof (such as (human) serum albumin or suitable fragments thereof) orto one or more binding units that can bind to serum proteins (such as,for example, domain antibodies, immunoglobulin single variable domainsthat are suitable for use as a domain antibody, single domainantibodies, immunoglobulin single variable domains that are suitable foruse as a single domain antibody, “dAb”'s, immunoglobulin single variabledomains that are suitable for use as a dAb, or Nanobodies that can bindto serum proteins such as serum albumin (such as human serum albumin),serum immunoglobulins such as IgG, or transferrine; reference is made tothe further description and references mentioned herein); polypeptidesin which an amino acid sequence of the invention is linked to an Fcportion (such as a human Fc) or a suitable part or fragment thereof; orpolypeptides in which the one or more immunoglobulin single variabledomains of the invention are suitable linked to one or more smallproteins or peptides that can bind to serum proteins (such as, withoutlimitation, the proteins and peptides described in WO 91/01743, WO01/45746, WO 02/076489, WO2008/068280, WO2009/127691).

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life e.g. in humans that is increased with more than 1hours, preferably more than 2 hours, more preferably more than 6 hours,such as more than 12 hours, or even more than 24, 48 or 72 hours,compared to the corresponding amino acid sequence of the invention perse.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life e.g. in humansthat is increased with more than 1 hours, preferably more than 2 hours,more preferably more than 6 hours, such as more than 12 hours, or evenmore than 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days).

In a particular preferred but non-limiting aspect of the invention, theinvention provides a polypeptide of the invention comprising i) oneCXCR7 binding immunoglobulin single variable domain as described herein;and ii) one or more (preferably one) serum albumin bindingimmunoglobulin single variable domain as described herein.

In a further preferred aspect, the invention provides a polypeptide ofthe invention comprising i) one CXCR7 binding immunoglobulin singlevariable domain as described herein; and ii) one or more (preferablyone) serum albumin binding immunoglobulin single variable domain of SEQID NO: 2 (Table B-1).

In a further preferred aspect, the invention provides a polypeptide ofthe invention comprising i) one CXCR7 binding immunoglobulin singlevariable domain as described herein; and ii) one or more (preferablyone) serum albumin binding immunoglobulin single variable domain withCDRs (defined according to the Kabat numbering) of SEQ ID NO: 2 (TableB-2, B-1).

Thus, for example, further reference (and thus incorporated byreference) is made in particular to the experimental part and furtherdescription of WO2008/068280, wherein further details on SEQ ID NO: 2 ismade and e.g. the half-life of a immunoglobulin single variable domainconstruct containing said sequence in rhesus monkeys is disclosed.

Generally, proteins or polypeptides that comprise or essentially consistof a single immunoglobulin single variable domain will be referred toherein as “monovalent” proteins or polypeptides or as “monovalentconstructs”. Proteins and polypeptides that comprise or essentiallyconsist of two or more immunoglobulin single variable domains (such asat least two immunoglobulin single variable domains of the invention orat least one immunoglobulin single variable domain of the invention andat least one other immunoglobulin single variable domain) will bereferred to herein as “multivalent” proteins or polypeptides or as“multivalent constructs”, and these may provide certain advantagescompared to the corresponding monovalent immunoglobulin single variabledomains of the invention. Some non-limiting examples of such multivalentconstructs will become clear from the further description herein.

According to another specific, but non-limiting aspect, a polypeptide ofthe invention comprises or essentially consists of at least oneimmunoglobulin single variable domain of the invention and at least oneother binding unit (i.e. directed against another epitope, antigen,target, protein or polypeptide), which is preferably also aimmunoglobulin single variable domain. Such proteins or polypeptides arealso referred to herein as “multispecific” proteins or polypeptides oras “multispecific constructs”, and these may comprise of twoimmunoglobulin single variable domains of the invention, such as oneimmunoglobulin single variable domain directed against CXCR7 and oneimmunoglobulin single variable domain against serum albumin. Suchmultispecific constructs will be clear to the skilled person based onthe disclosure herein; some preferred, but non-limiting examples of suchmultispecific immunoglobulin single variable domains are the constructsof SEQ ID NO's: 44 to 48 (see Table B-4).

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one immunoglobulin single variable domain of the invention,optionally one or more further immunoglobulin single variable domains,and at least one other amino acid sequence (such as a protein orpolypeptide) that confers at least one desired property to theimmunoglobulin single variable domain of the invention and/or to theresulting fusion protein. Again, such fusion proteins may providecertain advantages compared to the corresponding monovalentimmunoglobulin single variable domains of the invention such as e.g. mayprovide an increased half-life.

In the above constructs, the one or more immunoglobulin single variabledomains and/or other immunoglobulin single variable domains may bedirectly linked to each other and/or suitably linked to each other viaone or more linker sequences. Some suitable but non-limiting examples ofsuch linkers will become clear from the further description herein.

In one embodiment, the linker sequence joining the immunoglobulin singlevariable domains are SEQ ID NO: 49 to 58—see Table B-5, or a combinationof both, or as known in the art.

According to yet another specific, but non-limiting aspect, apolypeptide of the invention may for example be chosen from the groupconsisting of immunoglobulin single variable domains that have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more “sequence identity” (as defined herein) with one or more ofthe immunoglobulin single variable domains of SEQ ID NO's: 39 to 43 (seeTable B-3), in which the polypeptides are preferably as further definedherein, i.e. in the preferred format of one immunoglobulin singlevariable domain directed against CXCR7 and one immunoglobulin singevariable domain directed against serum albumin

According to yet another specific, but non-limiting aspect, apolypeptide of the invention may for example be chosen from the groupconsisting of polypeptides that have more than 80%, preferably more than90%, more preferably more than 95%, such as 99% or more “sequenceidentity” (as defined herein) with one or more of the polypeptides ofSEQ ID NO's: 44 to 48 (see Table B-4). Some illustrative non-limitingexamples of biparatopic and bispecific polypeptides of the invention aregiven in SEQ ID NO's: 78 to 89.

1.3. Compositions and Pharmaceutical Compositions of the Invention

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or composition comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc wherein which the parenteraladministration is preferred. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein. Such a pharmaceutical preparation orcomposition will generally be referred to herein as a “pharmaceuticalcomposition”. A pharmaceutical preparation or composition for use in anon-human organism will generally be referred to herein as a “veterinarycomposition”.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one polypeptide of the invention or at least one polypeptide ofthe invention and at least one suitable carrier, diluent or excipient(i.e., suitable for pharmaceutical use), and optionally one or morefurther active substances.

Generally, the polypeptides of the invention can be formulated andadministered in any suitable manner known per se. Reference is forexample made to the general background art cited above (and inparticular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 andWO 08/020079) as well as to the standard handbooks, such as Remington'sPharmaceutical Sciences, 18^(th) Ed., Mack Publishing Company, USA(1990), Remington, the Science and Practice of Pharmacy, 21th Edition,Lippincott Williams and Wilkins (2005); or the Handbook of TherapeuticAntibodies (S. Dubel, Ed.), Wiley, Weinheim, 2007 (see for example pages252-255).

The polypeptides of the invention may be formulated and administered inany manner known per se for conventional antibodies and antibodyfragments (including ScFv's and diabodies) and other pharmaceuticallyactive proteins. Such formulations and methods for preparing the samewill be clear to the skilled person, and for example includepreparations suitable for parenteral administration (for exampleintravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal,intra-arterial or intrathecal administration) or for topical (i.e.,transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, those mentioned onpage 143 of WO 08/020079. In one embodiment, the preparation is anaqueous solution or suspension.

The polypeptides of the invention can be administered using gene therapymethods of delivery. See, e.g., U.S. Pat. No. 5,399,346, which isincorporated by reference for its gene therapy delivery methods. Using agene therapy method of delivery, primary cells transfected with the geneencoding an amino acid sequence, polypeptide of the invention canadditionally be transfected with tissue specific promoters to targetspecific organs, tissue, grafts, tumors, or cells and can additionallybe transfected with signal and stabilization sequences for subcellularlylocalized expression.

Thus, the polypeptides of the invention may be systemicallyadministered, e.g., orally, in combination with a pharmaceuticallyacceptable vehicle such as an inert diluent or an assimilable ediblecarrier. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, thepolypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of thepolypeptide of the invention. Their percentage in the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 60% of the weight of a given unit dosage form. Theamount of the polypeptide of the invention in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

For local administration at the site of tumor resection, thepolypeptides of the invention may be used in biodegradable polymericdrug delivery systems, slow release poly(lactic-co-glycolic acidformulations and the like (Hart et al., Cochrane Database Syst Rev. 2008Jul. 16; (3): CD007294).

In a further preferred aspect of the invention, the polypeptides of theinvention, such as a polypeptide consisting essentially of onemonovalent anti-human CXCR7 immunoglobulin single variable domain and ofone monovalent anti-human serum albumin immunoglobulin single variabledomain linked by a GS linker, may have a beneficial distribution andkinetics profile in solid tumors compared to conventional antibodiessuch as e.g. IgG.

The tablets, troches, pills, capsules, and the like may also containbinders, excipients, disintegrating agents, lubricants and sweetening orflavoring agents, for example those mentioned on pages 143-144 of WO08/020079. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the polypeptides of the invention, sucrose orfructose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any unit dosage form should bepharmaceutically acceptable and substantially non-toxic in the amountsemployed. In addition, the polypeptides of the invention may beincorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The polypeptides of the invention may also be administered intravenouslyor intraperitoneally by infusion or injection. Particular examples areas further described on pages 144 and 145 of WO 08/020079.

For topical administration, the polypeptides of the invention may beapplied in pure form, i.e., when they are liquids. However, it willgenerally be desirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid. Particular examples are as furtherdescribed on page 145 of WO 08/020079.

Generally, the concentration of the polypeptides of the invention in aliquid composition, such as a lotion, will be from about 0.1-25 wt-%,preferably from about 0.5-10 wt-%. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the polypeptides of the invention required for use intreatment will vary not only with the particular polypeptide selectedbut also with the route of administration, the nature of the conditionbeing treated and the age and condition of the patient and will beultimately at the discretion of the attendant physician or clinician.Also the dosage of the polypeptides of the invention varies depending onthe target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one diseases and disorders associated withCXCR7, said method comprising administering, to a subject in needthereof, a pharmaceutically active amount of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with CXCR7, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signaling in which CXCR7 is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of a Polypeptide ofthe invention, of a polypeptide of the invention, and/or of apharmaceutical composition comprising the same. In one embodiment, theinvention relates to a method for the prevention and/or treatment of atleast one disease or disorder that can be treated by modulating CXCR7,its biological or pharmacological activity, and/or the biologicalpathways or signaling in which CXCR7 are involved, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of a polypeptide of the invention, and/orof a pharmaceutical composition comprising the same. In one embodiment,said pharmaceutically effective amount may be an amount that issufficient to modulate CXCR7, its biological or pharmacologicalactivity, and/or the biological pathways or signaling in which CXCR7 isinvolved; and/or an amount that provides a level of the polypeptide ofthe invention in the circulation that is sufficient to modulate CXCR7,its biological or pharmacological activity, and/or the biologicalpathways or signaling in which CXCR7 is involved.

In one embodiment the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can beprevented and/or treated by administering a polypeptide of theinvention, or a nucleotide construct of the invention encoding the same,and/or of a pharmaceutical composition comprising the same, to apatient. In one embodiment, the method comprises administering apharmaceutically active amount of a polypeptide of the invention, or anucleotide construct of the invention encoding the same, and/or of apharmaceutical composition comprising the same to a subject in needthereof.

In one embodiment the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can beprevented and/or treated by inhibiting binding of CXCL12 and/or CXCL11to CXCR7 in specific cells or in a specific tissue of a subject to betreated (and in particular, by inhibiting binding of CXCL12 and/orCXCL11 to CXCR7 in cancer cells or in a tumor present in the subject tobe treated), said method comprising administering a pharmaceuticallyactive amount of a polypeptide of the invention, or a nucleotideconstruct of the invention encoding the same, and/or of a pharmaceuticalcomposition comprising the same, to a subject in need thereof.

In one embodiment, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apolypeptide of the invention, or a nucleotide construct of the inventionencoding the same, and/or of a pharmaceutical composition comprising thesame.

In one embodiment, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of apolypeptide of the invention, or a nucleotide construct of the inventionencoding the same, and/or of a pharmaceutical composition comprising thesame.

In the above methods, the amino acid sequences, polypeptides of theinvention and/or the compositions comprising the same can beadministered in any suitable manner, depending on the specificpharmaceutical formulation or composition to be used. Thus, thepolypeptides of the invention and/or the compositions comprising thesame can for example be administered orally, intraperitoneally (e.g.intravenously, subcutaneously, intramuscularly, or via any other routeof administration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The polypeptides of the invention and/or the compositions comprising thesame are administered according to a regime of treatment that issuitable for preventing and/or treating the disease or disorder to beprevented or treated. The clinician will generally be able to determinea suitable treatment regimen, depending on factors such as the diseaseor disorder to be prevented or treated, the severity of the disease tobe treated and/or the severity of the symptoms thereof, the polypeptideof the invention to be used, the specific route of administration andpharmaceutical formulation or composition to be used, the age, gender,weight, diet, general condition of the patient, and similar factors wellknown to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more polypeptides of the invention, or of one or more compositionscomprising the same, in one or more pharmaceutically effective amountsor doses. The specific amount(s) or doses to administered can bedetermined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific polypeptide of theinvention to be used, the specific route of administration and thespecific pharmaceutical formulation or composition used, thepolypeptides of the invention will generally be administered in anamount between 1 gram and 0.01 microgram per kg body weight per day,preferably between 0.1 gram and 0.1 microgram per kg body weight perday, such as about 1, 10, 100 or 1000 microgram per kg body weight perday, either continuously (e.g. by infusion), as a single daily dose oras multiple divided doses during the day. The clinician will generallybe able to determine a suitable daily dose, depending on the factorsmentioned herein. It will also be clear that in specific cases, theclinician may choose to deviate from these amounts, for example on thebasis of the factors cited above and his expert judgment. Generally,some guidance on the amounts to be administered can be obtained from theamounts usually administered for comparable conventional antibodies orantibody fragments against the same target administered via essentiallythe same route, taking into account however differences inaffinity/avidity, efficacy, biodistribution, half-life and similarfactors well known to the skilled person.

In one embodiment, a single contiguous polypeptide of the invention willbe used. In one embodiment two or more polypeptides of the invention areprovided in combination.

The polypeptides of the invention may be used in combination with one ormore further pharmaceutically active compounds or principles, i.e., as acombined treatment regimen, which may or may not lead to a synergisticeffect. Again, the clinician will be able to select such furthercompounds or principles, as well as a suitable combined treatmentregimen, based on the factors cited above and his expert judgment.

In particular, the polypeptides of the invention may be used incombination with other pharmaceutically active compounds or principlesthat are or can be used for the prevention and/or treatment of thediseases and disorders cited herein, as a result of which a synergisticeffect may or may not be obtained. Examples of such compounds andprinciples, as well as routes, methods and pharmaceutical formulationsor compositions for administering them will be clear to the clinician,and generally include the cytostatic active principles usually appliedfor the treatment of the tumor to be treated.

Specific contemplated combinations for use with the polypeptides of theinvention for oncology include, but are not limited to, e.g., CXCR4antagonists such as e.g. AMD3100, other chemokine receptor antagonists,TAXOL® (paclitaxel); gemcitobine; cisplatin; cIAP inhibitors (such asinhibitors to cIAP1, cIAP2 and/or XIAP); MEK inhibitors including butnot limited to, e.g., U0126, PD0325901; bRaf inhibitors including butnot limited to, e.g., RAF265; and mTOR inhibitors including but notlimited to, e.g., RAD001; VEGF inhibitors including but not limited toe.g. bevacizumab, sutinib and sorafenib; Her 2 inhibitors including butnot limited to e.g. trastuzumab and lapatinib; PDGFR, FGFR, src, JAK,STAT and/or GSK3 inhibitors; selective estrogen receptor modulatorsincluding but not limited to tamoxifen; estrogen receptor downregulatorsincluding but not limited to fulvestrant. Specific contemplatedcombinations for use with the polypeptides of the invention forinflammatory conditions include, but are not limited to, e.g.,interferon beta 1 alpha and beta, natalizumab; TNF alpha antagonistsincluding but not limited to e.g. infliximab, adalimumab, certolizumabpegol, etanercept; disease-modifying antirheumatic drugs such as e.g.methotrexate (MTX); glucocortioids including but not limited to e.g.hydrocortisone; Nonsteroidal anti-inflammatory drugs including but notlimited to e.g. ibuprofen, sulindac.

Other specific compounds/polypeptides that could be used in combination(therapy) with the compounds/polypeptides of the invention are the aminoacid sequences and polypeptides directed against CXCR4 that aredescribed in the international application WO 09/138519 by Ablynx N.V.,the non-prepublished U.S. application 61/358,495 by Ablynx N.V. filed onJun. 25, 2010; the PCT application PCT/EP210/064766 by Ablynx N.V. filedon Oct. 4, 2010; and/or the PCT application PCT/EP2011/050156 by AblynxN.V. filed on Jan. 7, 2011.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of polypeptide ofthe invention in the preparation of a pharmaceutical composition forprevention and/or treatment of at least one diseases and disordersassociated with CXCR7; and/or for use in one or more of the methods oftreatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. In veterinaryapplications, the subject to be treated includes any animal raised forcommercial purposes or kept as a pet. As will be clear to the skilledperson, the subject to be treated will in particular be a personsuffering from, or at risk of, the diseases and disorders mentionedherein.

The invention relates to the use of a polypeptide of the invention, or anucleotide encoding the same, in the preparation of a pharmaceuticalcomposition for the prevention and/or treatment of at least one diseaseor disorder that can be prevented and/or treated by administering apolypeptide of the invention, or a nucleotide encoding the same, and/ora pharmaceutical composition of the same to a patient.

More in particular, the invention relates to the use of a polypeptide ofthe invention, or a nucleotide encoding the same, in the preparation ofa pharmaceutical composition for the prevention and/or treatment ofdiseases and disorders associated with CXCR7, and in particular for theprevention and treatment of one or more of the diseases and disorderslisted herein.

Again, in such a pharmaceutical composition, the one or more polypeptideof the invention, or nucleotide encoding the same, and/or apharmaceutical composition of the same, may also be suitably combinedwith one or more other active principles, such as those mentionedherein.

The invention also relates to a composition (such as, withoutlimitation, a pharmaceutical composition or preparation as furtherdescribed herein) for use, either in vitro (e.g. in an in vitro orcellular assay) or in vivo (e.g. in an a single cell or multicellularorganism, and in particular in a mammal, and more in particular in ahuman being, such as in a human being that is at risk of or suffers froma disease or disorder of the invention).

In the context of the present invention, “modulating” or “to modulate”generally means reducing or inhibiting the activity of CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1), as measured using a suitable invitro, cellular or in vivo assay (such as those mentioned herein). Inparticular, reducing or inhibiting the activity of CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1), as measured using a suitable invitro, cellular or in vivo assay (such as those mentioned herein), by atleast 1%, preferably at least 5%, such as at least 10% or at least 25%,for example by at least 50%, at least 60%, at least 70%, at least 80%,or 90% or more, compared to activity of CXCR7 and in particular humanCXCR7 (SEQ ID NO: 1) in the same assay under the same conditions butwithout the presence of the polypeptide of the invention.

Modulating may for example involve reducing or inhibiting the bindingCXCR7 to one of its substrates or ligands and/or competing with naturalligands (CXCL11 and/or CXCL12), substrate for binding to CXCR7.

1.4. Generation of the Polypeptides of the Invention

The invention further relates to methods for preparing or generating theimmunoglobulin single variable domains, polypeptides, nucleic acids,host cells, products and compositions described herein. Some preferredbut non-limiting examples of such methods will become clear from thefurther description herein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of immunoglobulin single    variable domains; and-   b) screening said set, collection or library of immunoglobulin    single variable domains for immunoglobulin single variable domains    that can bind to and/or have affinity for CXCR7 and in particular    human CXCR7 (SEQ ID NO: 1);    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1).

In such a method, the set, collection or library of immunoglobulinsingle variable domains may be any suitable set, collection or libraryof immunoglobulin single variable domains. For example, the set,collection or library of immunoglobulin single variable domains may be aset, collection or library of immunoglobulin sequences (as describedherein), such as a naïve set, collection or library of immunoglobulinsequences; a synthetic or semi-synthetic set, collection or library ofimmunoglobulin sequences; and/or a set, collection or library ofimmunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of immunoglobulinsingle variable domains may be a set, collection or library of heavy orlight chain variable domains (such as VL-, VH- or VHH domains). Forexample, the set, collection or library of immunoglobulin singlevariable domains may be a set, collection or library of domainantibodies or single domain antibodies, or may be a set, collection orlibrary of immunoglobulin single variable domains that are capable offunctioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofimmunoglobulin single variable domains may be an immune set, collectionor library of immunoglobulin sequences, for example derived from amammal that has been suitably immunized with CXCR7 and in particularhuman CXCR7 (SEQ ID NO: 1) or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

In the above methods, the set, collection or library of immunoglobulinsingle variable domains may be displayed on a phage, phagemid, ribosomeor suitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of)immunoglobulin single variable domains will be clear to the personskilled in the art, for example on the basis of the further disclosureherein. Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating immunoglobulin singlevariable domains comprises at least the steps of:

-   a) providing a collection or sample of cells expressing    immunoglobulin single variable domains;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for CXCR7 and in particular human CXCR7 (SEQ ID NO: 1);    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

In another aspect, the method for generating an amino acid sequencedirected against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) maycomprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding immunoglobulin single variable domains;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1);    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding immunoglobulin single variable domains may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of immunoglobulin sequences;a set, collection or library of nucleic acid sequences encoding asynthetic or semi-synthetic set, collection or library of immunoglobulinsequences; and/or a set, collection or library of nucleic acid sequencesencoding a set, collection or library of immunoglobulin sequences thathave been subjected to affinity maturation.

In another aspect, the method for generating an amino acid sequencedirected against CXCR7 and in particular human CXCR7 (SEQ ID NO: 1) maycomprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding immunoglobulin single variable domains;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1) and that is cross-blocked or    is cross blocking a immunoglobulin single variable domain or    polypeptide of the invention, e.g. SEQ ID NO: 39 to 43 (Table B-2);    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

The invention also relates to immunoglobulin single variable domainsthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid immunoglobulin sequence; and of expressing or synthesizing saidamino acid sequence in a manner known per se, such as by expression in asuitable host cell or host organism or by chemical synthesis.

Also, following the steps above, one or more immunoglobulin singlevariable domains of the invention may be suitably humanized, camilizedor otherwise sequence optimized (e.g. sequence optimized formanufacturability, stability and/or solubility); and/or the amino acidsequence(s) thus obtained may be linked to each other or to one or moreother suitable immunoglobulin single variable domains (optionally viaone or more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized, camilized or otherwisesequence optimized (e.g. sequence optimized for manufacturability,stability and/or solubility) and suitably expressed; and/or one or morenucleic acid sequences encoding an amino acid sequence of the inventionmay be linked to each other or to one or more nucleic acid sequencesthat encode other suitable immunoglobulin single variable domains(optionally via nucleotide sequences that encode one or more suitablelinkers), after which the nucleotide sequence thus obtained may besuitably expressed so as to provide a polypeptide of the invention.

The invention further relates to applications and uses of theimmunoglobulin single variable domains, compounds, constructs,polypeptides, nucleic acids, host cells, products and compositionsdescribed herein, as well as to methods for the diagnosis, preventionand/or treatment for diseases and disorders associated with CXCR7 and inparticular human CXCR7 (SEQ ID NO: 1). Some preferred but non-limitingapplications and uses will become clear from the further descriptionherein.

The invention also relates to the immunoglobulin single variabledomains, compounds, constructs, polypeptides, nucleic acids, host cells,products and compositions described herein for use in therapy.

In particular, the invention also relates to the immunoglobulin singlevariable domains, compounds, constructs, polypeptides, nucleic acids,host cells, products and compositions described herein for use intherapy of a disease or disorder that can be prevented or treated byadministering, to a subject in need thereof, of (a pharmaceuticallyeffective amount of) an amino acid sequence, compound, construct orpolypeptide as described herein.

More in particular, the invention relates to the immunoglobulin singlevariable domains, compounds, constructs, polypeptides, nucleic acids,host cells, products and compositions described herein for use intherapy of cancer.

1.5. Variants of Polypeptides and Immunoglobulin Single Variable Domainsof the Invention

Polypeptides of the invention and immunoglobulin single variable domains(that form part of the polypeptides of the invention) may be altered inorder to further improve potency or other desired properties.

Generally, an immunoglobulin single variable domain can be defined as apolypeptide with the formula 1FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table B-2 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) Immunoglobulin single variable domains ofthe invention. As will be clear to the skilled person, a combination ofCDR1, CDR2 and CDR3 sequences that occur in the same clone (i.e. CDR1,CDR2 and CDR3 sequences that are mentioned on the same line in TableB-2) will usually be preferred (although the invention in its broadestsense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences mentioned in Table B-2). Also, acombination of CDR sequences and framework sequences that occur in thesame clone (i.e. CDR sequences and framework sequences that arementioned on the same line in Table B-2) will usually be preferred(although the invention in its broadest sense is not limited thereto,and also comprises other suitable combinations of the CDR sequences andframework sequences mentioned in Table B-2, as well as combinations ofsuch CDR sequences and other suitable framework sequences, e.g. asfurther described herein).

Also, in the immunoglobulin single variable domains of the inventionthat comprise the combinations of CDR's mentioned in Table B-2, each CDRcan be replaced by a CDR chosen from the group consisting ofimmunoglobulin single variable domains that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity (as defined herein) with thementioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table B-2, a    conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table B-2;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table B-2.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table B-2 will generally be preferred.

Thus, in the immunoglobulin single variable domains of the invention, atleast one of the CDR1, CDR2 and CDR3 sequences present is suitablychosen from the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2; or from the group of CDR1, CDR2 andCDR3 sequences, respectively, that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% “sequence identity” (as defined herein) with at least one of theCDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-2; and/orfrom the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, that have 3, 2 or only 1 “amino acid difference(s)” (asdefined herein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the Nanobodies of theinvention bind to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1)with an affinity (suitably measured and/or expressed as a EC50 value, oralternatively as an IC₅₀ value, as further described herein in variousin vitro and/or in vivo potency or other assays) that is as definedherein.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence present is suitably chosen fromthe group consisting of the CDR3 sequences listed in Table B-2 or fromthe group of CDR3 sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the CDR3 sequences listed inTable B-2; and/or from the group consisting of the CDR3 sequences thathave 3, 2 or only 1 amino acid difference(s) with at least one of theCDR3 sequences listed in Table B-2.

Preferably, in the immunoglobulin single variable domains of theinvention, at least two of the CDR1, CDR2 and CDR3 sequences present aresuitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2 or from the groupconsisting of CDR1, CDR2 and CDR3 sequences, respectively, that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity with at least one of theCDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-2; and/orfrom the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, that have 3, 2 or only 1 “amino acid difference(s)” withat least one of the CDR1, CDR2 and CDR3 sequences, respectively, listedin Table B-2.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence present is suitably chosen fromthe group consisting of the CDR3 sequences listed in Table B-2 or fromthe group of CDR3 sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the CDR3 sequences listed inTable B-2, respectively; and at least one of the CDR1 and CDR2 sequencespresent is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table B-2 or from the group ofCDR1 and CDR2 sequences, respectively, that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of the CDR1and CDR2 sequences, respectively, listed in Table B-2; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table B-2.

Most preferably, in the immunoglobulin single variable domains of theinvention, all three CDR1, CDR2 and CDR3 sequences present are suitablychosen from the group consisting of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2 or from the group of CDR1, CDR2 andCDR3 sequences, respectively, that have at least 80%, preferably atleast 90%, more preferably at least 95%, even more preferably at least99% sequence identity with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2; and/or from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1,CDR2 and CDR3 sequences, respectively, listed in Table B-2.

Even more preferably, in the immunoglobulin single variable domains ofthe invention, at least one of the CDR1, CDR2 and CDR3 sequences presentis suitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2. Preferably, in thisaspect, at least one or preferably both of the other two CDR sequencespresent are suitably chosen from CDR sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of thecorresponding CDR sequences, respectively, listed in Table B-2; and/orfrom the group consisting of the CDR sequences that have 3, 2 or only 1amino acid difference(s) with at least one of the correspondingsequences, respectively, listed in Table B-2.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence present is suitably chosen fromthe group consisting of the CDR3 listed in Table B-2. Preferably, inthis aspect, at least one and preferably both of the CDR1 and CDR2sequences present are suitably chosen from the groups of CDR1 and CDR2sequences, respectively, that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with the CDR1 and CDR2 sequences, respectively, listedin Table B-2; and/or from the group consisting of the CDR1 and CDR2sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table B-2.

Even more preferably, in the immunoglobulin single variable domains ofthe invention, at least two of the CDR1, CDR2 and CDR3 sequences presentare suitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable B-2; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with at least one of thecorresponding sequences listed in Table B-2.

In particular, in the immunoglobulin single variable domains of theinvention, at least the CDR3 sequence is suitably chosen from the groupconsisting of the CDR3 sequences listed in Table B-2, and either theCDR1 sequence or the CDR2 sequence is suitably chosen from the groupconsisting of the CDR1 and CDR2 sequences, respectively, listed in TableB-2. Preferably, in this aspect, the remaining CDR sequence present issuitably chosen from the group of CDR sequences that have at least 80%,preferably at least 90%, more preferably at least 95%, even morepreferably at least 99% sequence identity with at least one of thecorresponding CDR sequences listed in Table B-2; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with the corresponding CDR sequences listed in Table B-2.

Even more preferably, in the immunoglobulin single variable domains ofthe invention, all three CDR1, CDR2 and CDR3 sequences present aresuitably chosen from the group consisting of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-2.

Also, generally, the combinations of CDR's listed in Table B-2 (i.e.those mentioned on the same line in Table B-2) are preferred. Thus, itis generally preferred that, when a CDR in a immunoglobulin singlevariable domain of the invention is a CDR sequence mentioned in TableB-2 or is suitably chosen from the group of CDR sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity with a CDR sequencelisted in Table B-2; and/or from the group consisting of CDR sequencesthat have 3, 2 or only 1 amino acid difference(s) with a CDR sequencelisted in Table B-2, that at least one and preferably both of the otherCDR's are suitably chosen from the CDR sequences that belong to the samecombination in Table B-2 (i.e. mentioned on the same line in Table B-1)or are suitably chosen from the group of CDR sequences that have atleast 80%, preferably at least 90%, more preferably at least 95%, evenmore preferably at least 99% sequence identity with the CDR sequence(s)belonging to the same combination and/or from the group consisting ofCDR sequences that have 3, 2 or only 1 amino acid difference(s) with theCDR sequence(s) belonging to the same combination. The other preferencesindicated in the above paragraphs also apply to the combinations ofCDR's mentioned in Table B-2.

Thus, by means of non-limiting examples, a polypeptide of the inventioncan for example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-2, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table B-2 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred immunoglobulin single variable domains of the inventionmay for example comprise: (1) a CDR1 sequence that has more than 80%sequence identity with one of the CDR1 sequences mentioned in Table B-2;a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of theCDR2 sequences mentioned in Table B-2 (but belonging to a differentcombination); and a CDR3 sequence that has more than 80% sequenceidentity with one of the CDR3 sequences mentioned in Table B-2 (butbelonging to a different combination); or (2) a CDR1 sequence that hasmore than 80% sequence identity with one of the CDR1 sequences mentionedin Table B-2; a CDR2 sequence, and one of the CDR3 sequences listed inTable B-2; or (3) a CDR1 sequence; a CDR2 sequence that has more than80% sequence identity with one of the CDR2 sequence listed in Table B-1;and a CDR3 sequence that has 3, 2 or 1 amino acid differences with theCDR3 sequence mentioned in Table B-2 that belongs to the samecombination as the CDR2 sequence.

Some particularly preferred immunoglobulin single variable domains ofthe invention may for example comprise: (1) a CDR1 sequence that hasmore than 80% sequence identity with one of the CDR1 sequences mentionedin Table B-2; a CDR2 sequence that has 3, 2 or 1 amino acid differencewith the CDR2 sequence mentioned in Table B-2 that belongs to the samecombination; and a CDR3 sequence that has more than 80% sequenceidentity with the CDR3 sequence mentioned in Table B-2 that belongs tothe same combination; (2) a CDR1 sequence; a CDR2 listed in Table B-2and a CDR3 sequence listed in Table B-2 (in which the CDR2 sequence andCDR3 sequence may belong to different combinations).

Some even more preferred immunoglobulin single variable domains of theinvention may for example comprise: (1) a CDR1 sequence that has morethan 80% sequence identity with one of the CDR1 sequences mentioned inTable B-2; the CDR2 sequence listed in Table B-1 that belongs to thesame combination; and a CDR3 sequence mentioned in Table B-2 thatbelongs to a different combination; or (2) a CDR1 sequence mentioned inTable B-2; a CDR2 sequence that has 3, 2 or 1 amino acid differenceswith the CDR2 sequence mentioned in Table B-2 that belongs to the samecombination; and a CDR3 sequence that has more than 80% sequenceidentity with the CDR3 sequence listed in Table B-2 that belongs to thesame or a different combination.

Particularly preferred immunoglobulin single variable domains of theinvention may for example comprise a CDR1 sequence mentioned in TableB-2, a CDR2 sequence that has more than 80% sequence identity with theCDR2 sequence mentioned in Table B-2 that belongs to the samecombination; and the CDR3 sequence mentioned in Table B-2 that belongsto the same combination.

In the most preferred immunoglobulin single variable domains of theinvention, the CDR1, CDR2 and CDR3 sequences present are suitably chosenfrom one of the combinations of CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-2.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa immunoglobulin single variable domain in which the CDR sequences (asdefined herein) have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more sequence identity (asdefined herein) with the CDR sequences of at least one of theimmunoglobulin single variable domains of SEQ ID NO's: 39 to 43 (seeTable B-3).

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the immunoglobulin single variable domains of SEQID NO's: 39 to 43 (see Table B-2), that comprise, compared to thecorresponding native V_(HH) sequence, at least one humanizingsubstitution (as defined herein), and in particular at least onehumanizing substitution in at least one of its framework sequences (asdefined herein).

It will be clear to the skilled person that the immunoglobulin singlevariable domains that are mentioned herein as “preferred” (or “morepreferred”, “even more preferred”, etc.) are also preferred (or morepreferred, or even more preferred, etc.) for use in the polypeptidesdescribed herein. Thus, polypeptides that comprise or essentiallyconsist of one or more “preferred” immunoglobulin single variabledomains of the invention will generally be preferred, and polypeptidesthat comprise or essentially consist of one or more “more preferred”immunoglobulin single variable domains of the invention will generallybe more preferred, etc.

1.6. Nucleotides, Host Cells of the Invention

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as a immunoglobulin singlevariable domain of the invention) or a polypeptide of the inventioncomprising the same. Again, as generally described herein for thenucleic acids of the invention, such a nucleic acid may be in the formof a genetic construct, as defined herein. Specific embodiments of thisaspect of the invention are provided in Table B-6, SEQ ID NO's: 59 to63.

In another preferred, but non-limiting aspect, the invention relates tonucleic acid sequences of immunoglobulin single variable domain in whichthe sequences (as defined herein) have more than 80%, preferably morethan 90%, more preferably more than 95%, such as 99% or more sequenceidentity (as defined herein) with the sequences of at least one ofnucleic acid sequence of the immunoglobulin single variable domains ofSEQ ID NO's: 59 to 63 (see Table B-6).

In another aspect, the invention relates to nucleic acid sequences thatcomprise the nucleic acid sequences of immunoglobulin single variabledomain in which the sequences (as defined herein) have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore sequence identity (as defined herein) with the sequences of atleast one of nucleic acid sequence of the immunoglobulin single variabledomains of SEQ ID NO's: 59 to 63 (see Table B-6).

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an amino acid sequence (suchas a immunoglobulin single variable domain) of the invention and/or apolypeptide of the invention comprising the same; and/or that contains anucleic acid of the invention. Some preferred but non-limiting examplesof such hosts or host cells will become clear from the furtherdescription herein.

As will be clear to the skilled person, one particularly useful methodfor preparing a polypeptide of the invention generally comprises thesteps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, polypeptide of the invention (also referred to herein    as a “nucleic acid of the invention”), optionally followed by:-   ii) isolating and/or purifying the polypeptide of the invention thus    obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one polypeptide of the invention;    optionally followed by:-   ii) isolating and/or purifying the polypeptide of the invention thus    obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the immunoglobulinsingle variable domains for the polypeptides of the invention givenherein, and/or can be isolated from a suitable natural source. Toprovide analogs, nucleotide sequences encoding naturally occurringV_(HH) domains can for example be subjected to site-directedmutagenesis, so at to provide a nucleic acid of the invention encodingsaid analog. Also, as will be clear to the skilled person, to prepare anucleic acid of the invention, also several nucleotide sequences, suchas at least one nucleotide sequence encoding a polypeptide of theinvention and for example nucleic acids encoding one or more linkers canbe linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring form of CXCR7 and in particular human CXCR7 (SEQ IDNO: 1) as a template. These and other techniques will be clear to theskilled person, and reference is again made to the standard handbooks,such as Sambrook et al. and Ausubel et al., mentioned above, as well asthe Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art and as described on pages 131-134 of WO 08/020079(incorporated herein by reference). Such genetic constructs generallycomprise at least one nucleic acid of the invention that is optionallylinked to one or more elements of genetic constructs known per se, suchas for example one or more suitable regulatory elements (such as asuitable promoter(s), enhancer(s), terminator(s), etc.) and the furtherelements of genetic constructs referred to herein. Such geneticconstructs comprising at least one nucleic acid of the invention willalso be referred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator; and optionally also-   iii) one or more further elements of genetic constructs known per    se;

in which the terms “operably connected” and “operably linked” have themeaning given on pages 131-134 of WO 08/020079; and in which the“regulatory elements”, “promoter”, “terminator” and “further elements”are as described on pages 131-134 of WO 08/020079; and in which thegenetic constructs may further be as described on pages 131-134 of WO08/020079.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the polypeptide of the invention.Suitable hosts or host cells will be clear to the skilled person, andmay for example be any suitable fungal, prokaryotic or eukaryotic cellor cell line or any suitable fungal, prokaryotic or eukaryotic organism,for example those described on pages 134 and 135 of WO 08/020079; aswell as all other hosts or host cells known per se for the expressionand production of antibodies and antibody fragments (including but notlimited to (single) domain antibodies and ScFv fragments), which will beclear to the skilled person. Reference is also made to the generalbackground art cited hereinabove, as well as to for example WO 94/29457;WO 96/34103; WO 99/42077.

The immunoglobulin single variable domains, and polypeptides of theinvention can for example also be produced in the milk of transgenicmammals, for example in the milk of rabbits, cows, goats or sheep (seefor example U.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S.Pat. No. 6,849,992 for general techniques for introducing transgenesinto mammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or turbers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the immunoglobulin single variable domains, andpolypeptides of the invention can also be expressed and/or produced incell-free expression systems, and suitable examples of such systems willbe clear to the skilled person. Some preferred, but non-limitingexamples include expression in the wheat germ system; in rabbitreticulocyte lysates; or in the E. coli Zubay system.

As mentioned above, one of the advantages of the use of immunoglobulinsingle variable domains is that the polypeptides based thereon can beprepared through expression in a suitable bacterial system, and suitablebacterial expression systems, vectors, host cells, regulatory elements,etc., will be clear to the skilled person, for example from thereferences cited above. It should however be noted that the invention inits broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of immunoglobulin single variable domains orimmunoglobulin single variable domain-containing protein therapeuticsinclude strains of E. coli, Pichia pastoris, S. cerevisiae that aresuitable for large scale expression/production/fermentation, and inparticular for large scale pharmaceutical (i.e. GMP grade)expression/production/fermentation. Suitable examples of such strainswill be clear to the skilled person. Such strains andproduction/expression systems are also made available by companies suchas Richter Helm (Hamburg, Germany) or CMC Biologics (Soeborg, Denmark).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a immunoglobulin singlevariable domain-containing recombinant protein for which glycosylationis desired or required would necessitate the use of mammalian expressionhosts that have the ability to glycosylate the expressed protein. Inthis respect, it will be clear to the skilled person that theglycosylation pattern obtained (i.e. the kind, number and position ofresidues attached) will depend on the cell or cell line that is used forthe expression. Preferably, either a human cell or cell line is used(i.e. leading to a protein that essentially has a human glycosylationpattern) or another mammalian cell line is used that can provide aglycosylation pattern that is essentially and/or functionally the sameas human glycosylation or at least mimics human glycosylation.Generally, prokaryotic hosts such as E. coli do not have the ability toglycosylate proteins, and the use of lower eukaryotes such as yeastusually leads to a glycosylation pattern that differs from humanglycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired polypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, thepolypeptide of the invention is glycosylated. According to anothernon-limiting aspect of the invention, the polypeptide of the inventionis non-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the polypeptide of the invention is produced in a bacterial cell, inparticular a bacterial cell suitable for large scale pharmaceuticalproduction, such as cells of the strains mentioned above.

According to another preferred, but non-limiting aspect of theinvention, the polypeptide of the invention is produced in a yeast cell,in particular a yeast cell suitable for large scale pharmaceuticalproduction, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the polypeptide of the invention is produced in a mammaliancell, in particular in a human cell or in a cell of a human cell line,and more in particular in a human cell or in a cell of a human cell linethat is suitable for large scale pharmaceutical production, such as thecell lines mentioned hereinabove.

As further described on pages 138 and 139 of WO 08/020079, whenexpression in a host cell is used to produce the immunoglobulin singlevariable domains, and the polypeptides of the invention, theimmunoglobulin single variable domains, and polypeptides of theinvention can be produced either intracellullarly (e.g. in the cytosol,in the periplasma or in inclusion bodies) and then isolated from thehost cells and optionally further purified; or can be producedextracellularly (e.g. in the medium in which the host cells arecultured) and then isolated from the culture medium and optionallyfurther purified. Thus, according to one non-limiting aspect of theinvention, the polypeptide of the invention is an amino acid sequence,polypeptide that has been produced intracellularly and that has beenisolated from the host cell, and in particular from a bacterial cell orfrom an inclusion body in a bacterial cell. According to anothernon-limiting aspect of the invention, the amino acid sequence, orpolypeptide of the invention is an amino acid sequence, or polypeptidethat has been produced extracellularly, and that has been isolated fromthe medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude those mentioned on pages 139 and 140 of WO 08/020079.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include those mentioned on page 140 of WO 08/020079.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), a polypeptide of the invention (and in case of a hostorganism: in at least one cell, part, tissue or organ thereof). Theinvention also includes further generations, progeny and/or offspring ofthe host cell or host organism of the invention, that may for instancebe obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the immunoglobulin single variabledomains of the invention, the transformed host cell or transformed hostorganism may generally be kept, maintained and/or cultured underconditions such that the (desired) amino acid sequence, or polypeptideof the invention is expressed/produced. Suitable conditions will beclear to the skilled person and will usually depend upon the hostcell/host organism used, as well as on the regulatory elements thatcontrol the expression of the (relevant) nucleotide sequence of theinvention. Again, reference is made to the handbooks and patentapplications mentioned above in the paragraphs on the genetic constructsof the invention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theimmunoglobulin single variable domains of the invention may be expressedin a constitutive manner, in a transient manner, or only when suitablyinduced.

It will also be clear to the skilled person that the amino acidsequence, or polypeptide of the invention may (first) be generated in animmature form (as mentioned above), which may then be subjected topost-translational modification, depending on the host cell/hostorganism used. Also, the amino acid sequence, or polypeptide of theinvention may be glycosylated, again depending on the host cell/hostorganism used.

The amino acid sequence, or polypeptide of the invention may then beisolated from the host cell/host organism and/or from the medium inwhich said host cell or host organism was cultivated, using proteinisolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, or polypeptide of the invention) and/or preparativeimmunological techniques (i.e. using antibodies against the amino acidsequence to be isolated).

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced herein.

The invention will now be further described by means of the followingnon-limiting preferred aspects, figures and examples:

Preferred Non-Limiting Aspects:

-   Aspect A-1: An immunoglobulin single variable domain that is    directed against and/or that can specifically bind to CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1).-   Aspect A-2: An immunoglobulin single variable domain according to    aspect A-1, that is in essentially isolated form.-   Aspect A-3: An immunoglobulin single variable domain according to    aspect A-1 or A-2, for administration to a subject, wherein said    immunoglobulin single variable domain does not naturally occur in    said subject.-   Aspect A-4: An immunoglobulin single variable domain that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²    moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or    less and more preferably 10⁻⁸ to 10⁻¹² moles/liter. Such an    immunoglobulin single variable domain may in particular be an    immunoglobulin single variable domain according to any of the    preceding aspects.-   Aspect A-5: An immunoglobulin single variable domain that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with a rate of association (k_(on)-rate) of between 10²    M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as    between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹. Such an immunoglobulin single    variable domain may in particular be an immunoglobulin single    variable domain according to any of the preceding aspects.-   Aspect A-6: An immunoglobulin single variable domain that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with a rate of dissociation (k_(off) rate) between 1 s⁻¹ and    10⁻⁶ s⁻¹, preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably    between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁴ s⁻¹ and 10⁻⁶ s⁻¹.    Such an immunoglobulin single variable domain may in particular be    an immunoglobulin single variable domain according to any of the    preceding aspects.-   Aspect A-7: An immunoglobulin single variable domain that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with an affinity less than 500 nM, preferably less than 200    nM, more preferably less than 10 nM, such as less than 500 pM. Such    an immunoglobulin single variable domain may in particular be an    immunoglobulin single variable domain according to any of the    preceding aspects.-   Aspect A-8: An immunoglobulin single variable domain that can    specifically displace SDF-1 and/or I-TAC (CXCL11 and/or CXCL12) on    CXCR7 and in particular on human CXCR7 (SEQ ID NO: 1) with an    average Ki of less than 500 nM, preferably less than 200 nM, more    preferably less than 10 nM, such as less than 1 nM and an average    SDF-1 and/or I-TAC displacement of 50% or more, more preferably of    75% or more, even more preferably of 80% or more. Such an average Ki    and/or average displacement value may be determined e.g. in an assay    as described in example 9 or 10.-   Aspect A-9: An immunoglobulin single variable domain that can    specifically displace SDF-1 and/or I-TAC(CXCL11 and/or CXCL12) on    CXCR7 and in particular on human CXCR7 (SEQ ID NO: 1) with an    average Ki of less than 20 nM and an average SDF-1 and/or I-TAC    displacement of 70% or more. Such an average Ki and/or average    displacement value may be determined e.g. in an assay as described    in example 9 or 10.-   Aspect A-10: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of 4    framework regions (FR1 to FR4 respectively) and 3 complementarity    determining regions (CDR1 to CDR3 respectively).-   Aspect A-11: An immunoglobulin single variable domain according to    any of the preceding aspects, that is an immunoglobulin sequence.-   Aspect A-12: An immunoglobulin single variable domain according to    any of the preceding aspects, that is a naturally occurring    immunoglobulin sequence (from any suitable species) or a synthetic    or semi-synthetic immunoglobulin sequence.-   Aspect A-13: An immunoglobulin single variable domain according to    any of the preceding aspects that is a humanized immunoglobulin    sequence, a camelized immunoglobulin sequence or an immunoglobulin    sequence that has been obtained by techniques such as affinity    maturation.-   Aspect A-14: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a light    chain variable domain sequence (e.g. a VL-sequence); or of a heavy    chain variable domain sequence (e.g. a VH-sequence).-   Aspect A-15: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a heavy    chain variable domain sequence that is derived from a conventional    four-chain antibody or that essentially consist of a heavy chain    variable domain sequence that is derived from heavy chain antibody.-   Aspect A-16: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a domain    antibody (or an immunoglobulin single variable domain that is    suitable for use as a domain antibody), of a single domain antibody    (or an immunoglobulin single variable domain that is suitable for    use as a single domain antibody), of a “dAb” (or an immunoglobulin    single variable domain that is suitable for use as a dAb) or of a    Nanobody (including but not limited to a VHH sequence).-   Aspect A-17: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a    Nanobody.-   Aspect A-18: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a    Nanobody that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin single variable domains of SEQ ID NO's: 1 to 22        of WO 2009/138519, in which for the purposes of determining the        degree of amino acid identity, the amino acid residues that form        the CDR sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-1.

-   Aspect A-19: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of an    immunoglobulin single variable domain that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin single variable domains of SEQ ID NO's: 39 to 43,        in which for the purposes of determining the degree of amino        acid identity, the amino acid residues that form the CDR        sequences are disregarded;

and in which:

-   -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table A-1.

-   Aspect A-20: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a    polypeptide that comprises of    -   i) A first immunoglobulin single variable domain that has at        least 80% amino acid identity with an immunoglobulin single        variable domain selected from the group of immunoglobulin single        variable domain having SEQ ID NO's: 39 to 43, in which for the        purposes of determining the degree of amino acid identity, the        amino acid residues that form the CDR sequences are disregarded;        and that comprises of    -   ii) A second immunoglobulin single variable domain that has at        least 80% amino acid identity with the immunoglobulin single        variable domain having SEQ ID NO: 2, in which for the purposes        of determining the degree of amino acid identity, the amino acid        residues that form the CDR sequences are disregarded; and        optionally also comprises    -   iii) A linker.

-   Aspect A-21: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a    humanized or otherwise sequence optimized immunoglobulin single    variable domain.

-   Aspect A-22: An immunoglobulin single variable domain according to    any of the preceding aspects, that in addition to the at least one    binding site for binding against CXCR7 and in particular human CXCR7    (SEQ ID NO: 1), contains one or more further binding sites for    binding against other antigens, proteins or targets.    CDR-Based Aspects

-   Aspect B-1: An immunoglobulin single variable domain that is    directed against and/or that can specifically bind CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1), and that comprises one or    more (preferably one) stretches of amino acid residues chosen from    the group consisting of:    -   a) the immunoglobulin single variable domains of SEQ ID NO's: 9        to 13;    -   b) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 9 to 13;    -   c) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 9 to 13;    -   d) the immunoglobulin single variable domains of SEQ ID NO's: 19        to 23;    -   e) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 19 to 23;    -   f) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 19 to 23;    -   g) the immunoglobulin single variable domains of SEQ ID NO's: 29        to 33;    -   h) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 29 to 33;    -   i) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 29 to 33;    -   or any suitable combination thereof.    -   Such an immunoglobulin single variable domain may in particular        be VHH or sequence optimized VHH such as humanized, stabilized        and/or solubilized VHH.

-   Aspect B-2: An immunoglobulin single variable domain according to    aspect B-1, in which at least one of said stretches of amino acid    residues forms part of the antigen binding site for binding against    CXCR7 and in particular human CXCR7 (SEQ ID NO: 1).

-   Aspect B-3: An immunoglobulin single variable domain sequence that    is directed against and/or that can specifically bind CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1) and that comprises two or more    stretches of amino acid residues chosen from the group consisting    of:    -   a) the immunoglobulin single variable domains of SEQ ID NO's: 9        to 13;    -   b) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 9 to 13;    -   c) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 9 to 13;    -   d) the immunoglobulin single variable domains of SEQ ID NO's: 19        to 23;    -   e) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 19 to 23;    -   f) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 19 to 23;    -   g) the immunoglobulin single variable domains of SEQ ID NO's: 29        to 33;    -   h) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 29 to 33;    -   i) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 29 to 33;    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the immunoglobulin single variable domains        according to a), b) or c), the second stretch of amino acid        residues corresponds to one of the immunoglobulin single        variable domains according to d), e), f), g), h) or i); (ii)        when the first stretch of amino acid residues corresponds to one        of the immunoglobulin single variable domains according to        d), e) or f), the second stretch of amino acid residues        corresponds to one of the immunoglobulin single variable domains        according to a), b), c), g), h) or i); or (iii) when the first        stretch of amino acid residues corresponds to one of the        immunoglobulin single variable domains according to g), h) or        i), the second stretch of amino acid residues corresponds to one        of the immunoglobulin single variable domains according to a),        b), c), d), e) or f).    -   Such an immunoglobulin single variable domain may in particular        be VHH or sequence optimized VHH such as humanized, stabilized        and/or solubilized VHH.

-   Aspect B-4: An immunoglobulin single variable domain according to    aspect B-3, in which the at least two stretches of amino acid    residues forms part of the antigen binding site for binding against    CXCR7 and in particular human CXCR7 (SEQ ID NO: 1).

-   Aspect B-5: An immunoglobulin single variable domain sequence that    is directed against and/or that can specifically bind CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1) and that comprises three or    more stretches of amino acid residues, in which the first stretch of    amino acid residues is chosen from the group consisting of:    -   a) the immunoglobulin single variable domains of SEQ ID NO's: 9        to 13;    -   b) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 9 to 13;    -   c) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 9 to 13;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   d) the immunoglobulin single variable domain of SEQ ID NO's: 19        to 23;    -   e) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 19 to 23;    -   f) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 19 to 23;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   g) the immunoglobulin single variable domains of SEQ ID NO's: 29        to 33;    -   h) immunoglobulin single variable domains that have at least 80%        amino acid identity with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 29 to 33;    -   i) immunoglobulin single variable domains that have 3, 2, or 1        amino acid difference with at least one of the immunoglobulin        single variable domains of SEQ ID NO's: 29 to 33.    -   Such an immunoglobulin single variable domain may in particular        be VHH or sequence optimized VHH such as humanized, stabilized        and/or solubilized VHH.

-   Aspect B-6: An immunoglobulin single variable domain according to    aspect B-5, in which the at least three stretches of amino acid    residues forms part of the antigen binding site for binding against    CXCR7 and in particular human CXCR7 (SEQ ID NO: 1).

-   Aspect B-7: An immunoglobulin single variable domain that is    directed against and/or that can specifically bind CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1) in which the CDR sequences of    said immunoglobulin single variable domain have at least 70% amino    acid identity, preferably at least 80% amino acid identity, more    preferably at least 90% amino acid identity, such as 95% amino acid    identity or more or even essentially 100% amino acid identity with    the CDR sequences of at least one of the immunoglobulin single    variable domains of SEQ ID NO's: 39 to 43.    -   Such an immunoglobulin single variable domain may in particular        be VHH or sequence optimized VHH such as humanized, stabilized        and/or solubilized VHH.

-   Aspect C-1: An immunoglobulin single variable domain or polypeptide    that is directed against CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) and that cross-blocks the binding of at least one of the    immunoglobulin single variable domains of SEQ ID NO's: 39 to 43 or    polypeptides of SEQ ID NO's: 44 to 48 to CXCR7 and in particular    human CXCR7 (SEQ ID NO: 1). Such an immunoglobulin single variable    domain may in particular be an immunoglobulin single variable domain    according to any of the aspects A-1 to A-22 and/or according to    aspects B-1 to B-7. Also, preferably, such an immunoglobulin single    variable domain is able to specifically bind to CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1).

-   Aspect C-2: An immunoglobulin single variable domain or polypeptide    that is directed against CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) and that is cross-blocked from binding to CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1) by at least one of the    immunoglobulin single variable domains of SEQ ID NO's: 39 to 43 or    polypeptides of SEQ ID NO's: 44 to 48. Such an immunoglobulin single    variable domain may in particular be an immunoglobulin single    variable domain according to any of the aspects A-1 to A-22 and/or    according to aspects B-1 to B-7. Also, preferably, such an    immunoglobulin single variable domain is able to specifically bind    to CXCR7 and in particular human CXCR7 (SEQ ID NO: 1).

-   Aspect C-3: An immunoglobulin single variable domain or polypeptide    according to any of aspects C-1 or C-2, wherein the ability of said    immunoglobulin single variable domain to cross-block or to be    cross-blocked is detected in a displacement assay (e.g. as described    in examples 9 and/or 10 below).

-   Aspect C-4: An immunoglobulin single variable domain or polypeptide    according to any of aspects C-1 to C-3 wherein the ability of said    immunoglobulin single variable domain to cross-block or to be    cross-blocked is detected in an ELISA assay.

-   Aspect D-1: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7 or C-1 to C-7, that is in essentially    isolated form.

-   Aspect D-2: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, and/or D1 for administration    to a subject, wherein said immunoglobulin single variable domain    does not naturally occur in said subject.

-   Aspect D-3: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, and/or D1 to D-2 that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²    moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or    less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.

-   Aspect D-4: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, and/or D-1 to D-3 that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with a rate of association (k_(on)-rate) of between 10²    M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as    between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.

-   Aspect D-5: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, and/or D-1 to D-4 that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with a rate of dissociation (k_(off) rate) between 1 s⁻¹ and    10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably    between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁴ s⁻¹ and 10⁻⁶ s⁻¹.

-   Aspect D-6: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, and/or D-1 to D-5 that can    specifically bind to CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) with an affinity less than 500 nM, preferably less than 200    nM, more preferably less than 10 nM, such as less than 500 pM.    -   The immunoglobulin single variable domains according to aspects        D-1 to D-6 may in particular be an immunoglobulin single        variable domain according to any of the aspects A-1 to A-22.

-   Aspect E-1: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7 and/or D1 to D-6, that is a    naturally occurring immunoglobulin single variable domain (from any    suitable species) or a synthetic or semi-synthetic immunoglobulin    single variable domain.

-   Aspect E-2: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 that is    sequence optimized

-   Aspect E-3: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or D-1 or D-2,    that is stabilized.

-   Aspect E-4: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-3,    that is a naturally occurring immunoglobulin sequence (from any    suitable species) or a synthetic or semi-synthetic immunoglobulin    sequence.

-   Aspect E-5: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-4    that is a humanized immunoglobulin sequence, a camelized    immunoglobulin sequence or an immunoglobulin sequence that has been    obtained by techniques such as affinity maturation.

-   Aspect E-6: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-5    that essentially consists of a light chain variable domain sequence    (e.g. a V_(L)-sequence); or of a heavy chain variable domain    sequence (e.g. a V_(H)-sequence).

-   Aspect E-7: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-6,    that essentially consists of a heavy chain variable domain sequence    that is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.

-   Aspect E-8: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-7,    that essentially consists of a domain antibody (or an immunoglobulin    single variable domain that is suitable for use as a domain    antibody), of a single domain antibody (or an immunoglobulin single    variable domain that is suitable for use as a single domain    antibody), of a “dAb” (or an immunoglobulin single variable domain    that is suitable for use as a dAb) or of a Nanobody (including but    not limited to a V_(HH) sequence).

-   Aspect E-9: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-8    that essentially consists of a Nanobody.

-   Aspect E-10: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-9    that essentially consists of a immunoglobulin single variable domain    that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin single variable domains described herein, in        which for the purposes of determining the degree of amino acid        identity, the amino acid residues that form the CDR sequences        are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.

-   Aspect E-11: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to    E-10, that essentially consists of an immunoglobulin single variable    domain that    -   i) has at least 80% amino acid identity with at least one of the        An immunoglobulin single variable domains of SEQ ID NO's: 39 to        43, in which for the purposes of determining the degree of amino        acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.

-   Aspect E-12: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to E-11    that essentially consists of a humanized immunoglobulin single    variable domain

-   Aspect E-13: An immunoglobulin single variable domain according to    any of the aspects B-1 to B-7, C-1 to C-7, D1 to D-6, and/or E-1 to    E-11, that in addition to the at least one binding site for binding    formed by the CDR sequences, contains one or more further binding    sites for binding against other antigens, proteins or targets.    -   The immunoglobulin single variable domains according to aspects        E-1 to E-13 may in particular be an immunoglobulin single        variable domain according to any of the aspects A-1 to A-22.        Polypeptides

-   Aspect K-1: Polypeptide that comprises of one or more (preferably    one) immunoglobulin single variable domains according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, and/or E-1    to E-13, and optionally further comprises one or more peptidic    linkers.

-   Aspect K-2: Polypeptide according to aspect K-1, which additionally    comprises one or more (preferably one) immunoglobulin single    variable domain directed against serum albumin

-   Aspect K-3: Polypeptide according to any of aspects K-1 or K-2, in    which said immunoglobulin single variable domain directed against    serum albumin is directed against human serum albumin

-   Aspect K-4: Polypeptide according to any of aspects K-1 to K-3, in    which said one or more immunoglobulin single variable domain    directed against serum albumin is an immunoglobulin single variable    domain with SEQ ID NO: 2.

-   Aspect K-5: Polypeptide that comprises of one or more (preferably    one) immunoglobulin single variable domains according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, and/or E-1    to E-13, one or more cytotoxic payloads, and optionally further    comprises one or more peptidic linkers.

-   Aspect K-6: Polypeptide that comprises or essentially consists of    one or more (preferably one) immunoglobulin single variable domains    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, and/or E-1 to E-13, one or more (and preferably one)    immunoglobulin single variable domains (preferably Nanobody)    directed against CXCR4 and optionally further comprises one or more    peptidic linkers.

-   Aspect K-7: Polypeptide that comprises or essentially consists of at    least one (preferably one) immunoglobulin single variable domain    (preferably Nanobody) directed against (human) CXCR7 and at least    one (cyto)toxic group, moiety or payload (optionally linked    chemically or via one or more suitable linkers or spacers).

-   Aspect K-8: Polypeptide that comprises or essentially consists of at    least one (preferably one) immunoglobulin single variable domain    (preferably Nanobody) directed against (human) CXCR7, at least one    (preferably one) immunoglobulin single variable domain (preferably    Nanobody) directed against (human) CXCR4 and at least one    (cyto)toxic group, moiety or payload (optionally linked chemically    or via one or more suitable linkers or spacers).

-   Aspect K-9: Polypeptide that comprises or essentially consists of at    least one (preferably one) immunoglobulin single variable domain    (preferably Nanobody) directed against (human) CXCR7 and at least    one (preferably one) immunoglobulin single variable domain    (preferably Nanobody) directed against (human) CXCR4 (optionally    linked chemically or via one or more suitable linkers or spacers).

-   Aspect K-10: Polypeptide that comprises or essentially consists of    at least one (preferably one) immunoglobulin single variable domain    (preferably Nanobody) directed against (human) CXCR7, at least one    (preferably one) immunoglobulin single variable domain (preferably    Nanobody) directed against (human) CXCR4, and a peptide or    immunoglobulin single variable domain (preferably Nanobody) directed    against (human) serum albumin (optionally linked chemically or via    one or more suitable linkers or spacers).

-   Aspect K-11: Polypeptide that comprises or essentially consists of    two immunoglobulin single variable domains (preferably Nanobody)    directed against (human) CXCR7, which are the same (optionally    linked chemically or via one or more suitable linkers or spacers).

-   Aspect K-12: Polypeptide that comprises or essentially consists of    two immunoglobulin single variable domains (preferably Nanobody)    directed against (human) CXCR7, which are different from each other    (optionally linked chemically or via one or more suitable linkers or    spacers).

-   Aspect K-13: Polypeptide that comprises or essentially consists of    two immunoglobulin single variable domains (preferably Nanobody)    directed against (human) CXCR7, which are the same, and a peptide or    immunoglobulin single variable domain (preferably Nanobody) directed    against (human) serum albumin (optionally linked chemically or via    one or more suitable linkers or spacers).

-   Aspect K-14: Polypeptide that comprises or essentially consists of    two immunoglobulin single variable domains (preferably Nanobody)    directed against (human) CXCR7, which are different from each other,    and a peptide or immunoglobulin single variable domain (preferably    Nanobody) directed against (human) serum albumin (optionally linked    chemically or via one or more suitable linkers or spacers).    Nucleic Acid

-   Aspect M-1: Nucleic acid or nucleotide sequence, that encodes an    immunoglobulin single variable domain according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, a    polypeptide according to any of aspects K-1 to K-4.

-   Aspect M-2: Nucleic acid or nucleotide sequence with SEQ ID NOs:    59-63 and 73-77 (Table B-6).    Host Cell

Aspect N-1: Host or host cell that expresses, or that under suitablecircumstances is capable of expressing, an immunoglobulin singlevariable domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1to C-4, D-1 to D-6, E-1 to E-13, a polypeptide according to any ofaspects K-1 to K-4; and/or that comprises a nucleic acid or nucleotidesequence according to aspect M-1 or M-2.

Compositions

-   Aspect O-1: Composition comprising at least one immunoglobulin    single variable domain according to any of aspects A-1 to A-22, B-1    to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, or at least one    polypeptide according to any of aspects K-1 to K-4, or nucleic acid    or nucleotide sequence according to aspects M-1 or M-2.-   Aspect O-2: Composition according to aspect O-1, which is a    pharmaceutical composition.-   Aspect O-3: Composition according to aspect O-2, which is a    pharmaceutical composition, that further comprises at least one    pharmaceutically acceptable carrier, diluent or excipient and/or    adjuvant, and that optionally comprises one or more further    pharmaceutically active polypeptides and/or compounds.    Making of Agent and Composition of the Invention-   Aspect P-1: Method for producing an immunoglobulin single variable    domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to    C-4, D-1 to D-6, E-1 to E-13, a polypeptide according to any of    aspects K-1 to K-4, said method at least comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect M-1, or aspect M-2;    -   optionally followed by:    -   b) isolating and/or purifying the immunoglobulin single variable        domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1        to C-4, D-1 to D-6, E-1 to E-13, a polypeptide according to any        of aspects K-1 to K-4.-   Aspect P-2: Method for producing an immunoglobulin single variable    domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to    C-4, D-1 to D-6, E-1 to E-13, a polypeptide according to any of    aspects K-1 to K-4, said method at least comprising the steps of:    -   a) cultivating and/or maintaining a host or host cell according        to aspect . . . under conditions that are such that said host or        host cell expresses and/or produces at least one immunoglobulin        single variable domain according to any of aspects A-1 to A-22,        B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, a polypeptide        according to any of aspects K-1 to K-4;    -   optionally followed by:    -   b) isolating and/or purifying the immunoglobulin single variable        domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1        to C-4, D-1 to D-6, E-1 to E-13, a polypeptide according to any        of aspects K-1 to K-4.        Method of Screening-   Aspect Q-1: Method for screening immunoglobulin single variable    domains directed against CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) that comprises at least the steps of:    -   a) providing a set, collection or library of nucleic acid        sequences encoding immunoglobulin single variable domains;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an        immunoglobulin single variable domain that can bind to and/or        has affinity for CXCR7 and in particular human CXCR7 (SEQ ID        NO: 1) and that is cross-blocked or is cross blocking a Nanobody        of the invention, e.g. SEQ ID NO: 39 to 43 (Table-B-3), or a        polypeptide or construct of the invention, e.g. SEQ ID NO: 44 to        48 (see Table B-4); and    -   c) isolating said nucleic acid sequence, followed by expressing        said immunoglobulin single variable domain.        Use of Agents of the Invention-   Aspect R-1: Method for the prevention and/or treatment of cancer and    of inflammatory diseases (such as e.g. mentioned herein), said    method comprising administering, to a subject in need thereof, a    pharmaceutically active amount of at least one immunoglobulin single    variable domain according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, a polypeptide according to any    of aspects K-1 to K-4; or composition according to aspect O-2 or    O-3.-   Aspect R-2: Method for the prevention and/or treatment of at least    one disease or disorder that is associated with CXCR7 and in    particular human CXCR7 (SEQ ID NO: 1), with its biological or    pharmacological activity, and/or with the biological pathways or    signalling in which CXCR7 and in particular human CXCR7 (SEQ ID    NO: 1) is involved, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one immunoglobulin single variable domain according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, a polypeptide according to any of aspects K-1 to K-4; or    composition according to aspect O-2 or O-3.-   Aspect R-3: Method for the prevention and/or treatment of at least    one disease or disorder that can be prevented and/or treated by    administering, to a subject in need thereof, at least one    immunoglobulin single variable domain according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, a    polypeptide according to any of aspects K-1 to K-4; or composition    according to aspect O-2 or O-3, said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one at least one immunoglobulin single    variable domain according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, a polypeptide according to any    of aspects K-1 to K-4; or composition according to aspect O-2 or    O-3.-   Aspect R-4: Method for immunotherapy, said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one immunoglobulin single variable domain    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, a polypeptide according to any of aspects K-1    to K-4; or composition according to aspect O-2 or O-3.-   Aspect R-5: An immunoglobulin single variable domain according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, a polypeptide according to any of aspects K-1 to K-4, a    pharmaceutical composition according to aspect O-2 or O-3 for use in    one or more of the methods according to aspects R-1 to R-3.-   Aspect R-6: A polypeptide according to any of aspects K-1 to K-4,    for the diagnosis, prevention and/or treatment of cancer.    Further Aspects:-   1. An immunoglobulin single variable domain that can specifically    displace SDF-1 and/or I-TAC on human CXCR7 (SEQ ID NO: 1) with an    average Ki of less than 1000 nM, more preferably less than 500 nM,    more preferably less than 100 nM, even more preferably less than 50    nM, most preferably less than 10 nM and an average SDF-1 and/or    I-TAC displacement of 60% to 80% or more (in the radioligand assay    as e.g. outlined in example 9) or 80% to 100% (in the FACS    competition assay as e.g. outlined in example 10).-   2. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID NO:            9,        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 9,        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 9,    -   and wherein CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domain of SEQ ID NO:            19;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 19;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 19;    -   and wherein CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID NO:            29;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 29;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 29.-   3. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID NO:            10,        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 10,        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 10,    -   and wherein CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domain of SEQ ID NO:            20;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 20;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 20;    -   and wherein CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID NO:            30;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 30;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 30.-   4. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID NO:            11,        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 11,        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 11,    -   and wherein CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domain of SEQ ID NO:            21;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 21;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 21;    -   and wherein CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID NO:            31;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 31;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 31.-   5. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID NO:            12,        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 12,        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 12,    -   and wherein CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domain of SEQ ID NO:            22;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 22;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 22;    -   and wherein CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID NO:            32;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 32;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 32.-   6. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID NO:            13,        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 13,        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 13,    -   and wherein CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domain of SEQ ID NO:            23;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 23;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 23;    -   and wherein CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID NO:            33;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 33;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 33.-   7. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID NO:            14,        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 14,        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 14,    -   and wherein CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domain of SEQ ID NO:            24;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 24;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 24;    -   and wherein CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID NO:            34;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 34;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO: 34.-   8. The immunoglobulin single variable domain according to any of    aspects 2 to 7, wherein the framework regions (FRs) have a sequence    identity of more than 80% (more preferably 85%, even more preferably    90%, most preferred 95%) with the FRs of SEQ ID NOs: 4 to 8 (FR1),    14 to 18 (FR2), 24 to 28 (FR3), 34 to 38 (FR4).-   9. A polypeptide comprising an immunoglobulin single variable domain    of any of aspects 1 to 8.-   10. The polypeptide according to aspect 9, wherein the polypeptide    is selected from the group consisting of polypeptides that have an    amino acid sequence with a sequence identity of more than 80% (more    preferably 85%, even more preferably 90%, most preferred 95%) with    the of SEQ ID NOs: 39 to 43.-   11. The polypeptide according to any of aspects 9 to 10 and    additionally comprising an immunoglobulin single variable domain    that binds human serum albumin such as e.g. Alb8 (SEQ ID NO: 2).-   12. The polypeptides according to any of aspects 9 to 11, wherein    the polypeptide is selected from the group consisting of    polypeptides that have an amino acid sequence with a sequence    identity of more than 80% (more preferably 85%, even more preferably    90%, most preferred 95%) with the of SEQ ID NOs: 44 to 48.-   13. A nucleic acid sequence encoding i) for an immunoglobulin single    variable domain according to any of aspects 1 to 8; or ii) for a    polypeptide according to any of aspects 9 to 12.-   14. A pharmaceutical composition comprising i) an immunoglobulin    single variable domain according to any of aspects 1 to 8; or ii) a    polypeptide according to any of aspects 9 to 12; and optionally a    pharmaceutically acceptable excipient.-   15. An immunoglobulin single variable domain according to any of    aspects 1 to 8 or a polypeptide according to any of aspects 9 to 12    for use in cancer.-   16. Method for producing an immunoglobulin single variable domain    according to any of aspects 1 to 9 or a polypeptide according to any    of aspects 9 to 12, said method at least comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect 13;        -   optionally followed by:    -   b) isolating and/or purifying the immunoglobulin single variable        domain according to any of aspects 1 to 9 or a polypeptide        according to any of aspects 9 to 12.-   17. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is the immunoglobulin single variable domains        of SEQ ID NO: 9,    -   and wherein CDR2 is the immunoglobulin single variable domain of        SEQ ID NO: 19;    -   and wherein CDR3 is the immunoglobulin single variable domains        of SEQ ID NO: 29.-   18. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is the immunoglobulin single variable domains        of SEQ ID NO: 10,    -   and wherein CDR2 is the immunoglobulin single variable domain of        SEQ ID NO: 20;    -   and wherein CDR3 is the immunoglobulin single variable domains        of SEQ ID NO: 30.-   19. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is the immunoglobulin single variable domains        of SEQ ID NO: 11,    -   and wherein CDR2 is the immunoglobulin single variable domain of        SEQ ID NO: 21;    -   and wherein CDR3 is the immunoglobulin single variable domains        of SEQ ID NO: 31.-   20. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is the immunoglobulin single variable domains        of SEQ ID NO: 12,    -   and wherein CDR2 is the immunoglobulin single variable domain of        SEQ ID NO: 22;    -   and wherein CDR3 is the immunoglobulin single variable domains        of SEQ ID NO: 32.-   21. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is the immunoglobulin single variable domains        of SEQ ID NO: 13,    -   and wherein CDR2 is the immunoglobulin single variable domain of        SEQ ID NO: 23;    -   and wherein CDR3 is the immunoglobulin single variable domains        of SEQ ID NO: 33.-   22. The immunoglobulin single variable domain of aspect 1, wherein    the immunoglobulin single variable domain comprises an amino acid    sequence with the formula 1    FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1);    -   wherein FR1 to FR4 refer to framework regions 1 to 4 and are        framework regions of an immunoglobulin single variable domain;    -   and wherein CDR1 is the immunoglobulin single variable domains        of SEQ ID NO: 14,    -   and wherein CDR2 is the immunoglobulin single variable domain of        SEQ ID NO: 24;    -   and wherein CDR3 is the immunoglobulin single variable domains        of SEQ ID NO: 34.

EXPERIMENTAL PART Sequences

TABLE B-1 Prior art sequences SEQ ID Name NO: Amino acid sequencesHuman CXCR7  1 MDLHLFDYSEPGNFSDISWPCNSSDCIVVDTV or hCXCR7MCPNMPNKSVLLYTLSFIYIFIFVIGMIANSVVV WVNIQAKTTGYDTHCYILNLAIADLWVVLTIPVWVVSLVQHNQWPMGELTCKVTHLIFSINLFG SIFFLTCMSVDRYLSITYFTNTPSSRKKMVRRVVCILVWLLAFCVSLPDTYYLKTVTSASNNETY CRSFYPEHSIKEWLIGMELVSVVLGFAVPFSIIAVFYFLLARAISASSDQEKHSSRKIIFSYVVVFLV CWLPYHVAVLLDIFSILHYIPFTCRLEHALFTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKA FIFKYSAKTGLTKLIDASRVSETEYSALEQSTK Alb8 2 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFG MSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSS Mouse CXCR7  3MDVHLFDYAEPGNYSDINWPCNSSDCIVVDTV or mCXCR7QCPTMPNKNVLLYTLSFIYIFIFVIGMIANSVVV WVNIQAKTTGYDTHCYILNLAIADLWVVITIPVWVVSLVQHNQWPMGELTCKITHLIFSINLFGSI FFLACMSVDRYLSITYFTGTSSYKKKMVRRVVCILVWLLAFFVSLPDTYYLKTVTSASNNETYCR SFYPEHSIKEWLIGMELVSVILGFAVPFTIIAIFYFLLARAMSASGDQEKHSSRKIIFSYVVVFLVCW LPYHFVVLLDIFSILHYIPFTCQLENVLFTALHVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIF KYSAKTGLTKLIDASRVSETEYSALEQNTK Tag-171 AAAHHHHHHGAAEQKLISEEDLNGAA Tag-2 72 AAAEQKLISEEDLNGAAHHHHHHCynomoglous 90 MDLHVFDYSEPGNFSDISWPCNSSDCIVVDTV CXCR7 orMCPNMPNKSVLLYTLAFIYIFIFVIGMIANSVVV cCXCR7WVNIQAKTTGYDTHCYILNLAIADLWVVLTIP VWVVSLVQHNQWPMGELTCKVTHLIFSINLFGSIFFLTCMSVDRYLSITYFTNTSSSRKKMVRRV VCVLVWLLAFCVSLPDTYYLKTVTSASNNETYCRSFYPEHSIKEWLIGMELVSVVLGFAVPFSVIA VFYFLLARAISASGDQEKHSSRKIIFSYVVVFLVCWLPYHVAVLLDIFSILHYIPFTCRLEHALFTAL HVTQCLSLVHCCVNPVLYSFINRNYRYELMKAFIFKYSAKTGLTKLIDASRVSETEYSALEQSTK

TABLE B-2Sequences for CDRs and frameworks, plus preferred combinations as provided infor formula I, namely FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Clone ID FR1 IDCDR1 ID FR2 07B11 4 EVQLVESGGNLVQAGGSLGL 9 IHIMG 14 WYRQAPGKQRDLSCAASVSISS VA 07C3 5 EVQLVESGGGLVQAGESLTLS 10 AYIMG 15 WFRQAPGKEREFCAASGRTLS VA 08A5 6 EVQLVESGGGLVQAGDSLRL 11 NYDMG 16 WFRQAPGKEREFSCAASGLTFS VG 08A10 7 EVQLVESGGGLVQAGGSLRL 12 IAAMG 17 WYRQATGKQRELSCAASGSIFS VA 14G3 8 EVQLVESGGGLVQPGGSLRIS 13 INYMG 18 WYRQAPGKQRELCAASGSIYL VA Alb8 64 EVQLVESGGGLVQPGNSLRLS 65 SFGMS 66 WVRQAPGKGLEWCAASGFTFS VS Clone ID CDR2 ID FR3 07B11 19 TITSGGSTAYADSVKG 24RFTVSKDNAKNTVYLQMDSLKPEDTS VYYCAA 07C3 20 GIWSGGYTHLADSAK 25RFSISRDNAKNTVYLQMNGLKPEDTA G VYYCAA 08A5 21 ASWWSGGAPYYSDSV 26RFTISRDNAKNTVYLQANSLRPEDTAV KG YYCAA 08A10 22 TITDGGTTTYADSVKG 27RVTISRDRSANTVYLAMNNLKPDDTA VYYCYA 14G3 23 TLTSGGSTNYAGSVKG 28RFAISRDNAKNTVYLQMNSLKPEDTA VYYCNI Alb8 67 SISGSGSDTLYADSVK 68RFTISRDNAKTTLYLQMNSLRPEDTAV G YYCTI Clone ID CDR3 ID FR4 07B11 29EVRNGVFGKWNHY 34 WGQGTQVTVSS 07C3 30 GLRGRQYSN 35 WGQGTQVTVSS 08A5 31KRLRSFASGGSYDY 36 WGQGTQVTVSS 08A10 32 YLRYTSRVPGDNY 37 WGQGTQVTVSS 14G333 GGTLYDRRRFES 38 WGQGTQVTVSS Alb8 69 GGSLSR 70 SSQGTLVTVSS (thefollowing terms: “ID” refers to the given SEQ ID NO. Preferredcombination of FR and CDR sequences for each NB construct are usedinterchangeably through-out the application)

TABLE B-3 Amino acid sequences of immunoglobulin singlevariable sequences of the invention Name of SEQ clone ID NO:Amino acid sequences 07B11 39 EVQLVESGGNLVQAGGSLGLSCAASVSISSIHIMGWYRQAPGKQRDLVATITSGGSTAYADSVK GRFTVSKDNAKNTVYLQMDSLKPEDTSVYYCAAEVRNGVFGKWNHYWGQGTQVTVSS 07C3 40 EVQLVESGGGLVQAGESLTLSCAASGRTLSAYIMGWFRQAPGKEREFVAGIWSGGYTHLADSAK GRFSISRDNAKNTVYLQMNGLKPEDTAVYYCAAGLRGRQYSNWGQGTQVTVSS 08A5 41 EVQLVESGGGLVQAGDSLRLSCAASGLTFSNYDMGWFRQAPGKEREFVGASWWSGGAPYYSD SVKGRFTISRDNAKNTVYLQANSLRPEDTAVYYCAAKRLRSFASGGSYDYWGQGTQVTVSS 08A10 42 EVQLVESGGGLVQAGGSLRLSCAASGSIFSIAAMGWYRQATGKQRELVATITDGGTTTYADSVK GRVTISRDRSANTVYLAMNNLKPDDTAVYYCYAYLRYTSRVPGDNYWGQGTQVTVSS 14G3 43 EVQLVESGGGLVQPGGSLRISCAASGSIYLINYMGWYRQAPGKQRELVATLTSGGSTNYAGSVK GRFAISRDNAKNTVYLQMNSLKPEDTAVYYCNIGGTLYDRRRFESWGQGTQVTVSS

TABLE B-4 Polypeptide sequences of the invention SEQ ID Name of cloneNO: Amino acid sequences 07B11-9GS-Alb8 44EVQLVESGGNLVQAGGSLGLSCAASVSISSIHI MGWYRQAPGKQRDLVATITSGGSTAYADSVKGRFTVSKDNAKNTVYLQMDSLKPEDTSVYYC AAEVRNGVFGKWNHYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGF TFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSS 07C3-9GS-Alb845 EVQLVESGGGLVQAGESLTLSCAASGRTLSAYI MGWFRQAPGKEREFVAGIWSGGYTHLADSAKGRFSISRDNAKNTVYLQMNGLKPEDTAVYYCA AGLRGRQYSNWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFG MSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSS 08A5-9GS-Alb8 46EVQLVESGGGLVQAGDSLRLSCAASGLTFSNY DMGWFRQAPGKEREFVGASWWSGGAPYYSDSVKGRFTISRDNAKNTVYLQANSLRPEDTAVY YCAAKRLRSFASGGSYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAAS GFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPE DTAVYYCTIGGSLSRSSQGTLVTVSS08A10-9GS-Alb8 47 EVQLVESGGGLVQAGGSLRLSCAASGSIFSIAAMGWYRQATGKQRELVATITDGGTTTYADSVK GRVTISRDRSANTVYLAMNNLKPDDTAVYYCYAYLRYTSRVPGDNYWGQGTQVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 14G3-9GS-Alb8 48EVQLVESGGGLVQPGGSLRISCAASGSIYLINY MGWYRQAPGKQRELVATLTSGGSTNYAGSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCN IGGTLYDRRRFESWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSF GMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC TIGGSLSRSSQGTLVTVSS 07B11-9GS-07C3 78EVQLVESGGNLVQAGGSLGLSCAASVSISSIHI MGWYRQAPGKQRDLVATITSGGSTAYADSVKGRFTVSKDNAKNTVYLQMDSLKPEDTSVYYC AAEVRNGVFGKWNHYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGESLTLSCAASGR TLSAYIMGWFRQAPGKEREFVAGIWSGGYTHLADSAKGRFSISRDNAKNTVYLQMNGLKPEDTA VYYCAAGLRGRQYSNWGQGTQVTVSS07C3-9GS-0B11 79 EVQLVESGGGLVQAGESLTLSCAASGRTLSAYIMGWFRQAPGKEREFVAGIWSGGYTHLADSAK GRFSISRDNAKNTVYLQMNGLKPEDTAVYYCAAGLRGRQYSNWGQGTQVTVSSGGGGSGGGSE VQLVESGGNLVQAGGSLGLSCAASVSISSIHIMGWYRQAPGKQRDLVATITSGGSTAYADSVKG RFTVSKDNAKNTVYLQMDSLKPEDTSVYYCAAEVRNGVFGKWNHYWGQGTQVTVSS 07B11-9GS-Alb8- 80EVQLVESGGNLVQAGGSLGLSCAASVSISSIHI 9GS-07C3MGWYRQAPGKQRDLVATITSGGSTAYADSVK GRFTVSKDNAKNTVYLQMDSLKPEDTSVYYCAAEVRNGVFGKWNHYWGQGTQVTVSSGGGG SGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTL YADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQAGESLTLSCAASGRTLSAYIMGWFRQAPGKEREFVAGIWSGGYTHLADSAK GRFSISRDNAKNTVYLQMNGLKPEDTAVYYCAAGLRGRQYSNWGQGTQVTVSS 07B11-9GS-07C3- 81EVQLVESGGNLVQAGGSLGLSCAASVSISSIHI 9GS-Alb8MGWYRQAPGKQRDLVATITSGGSTAYADSVK GRFTVSKDNAKNTVYLQMDSLKPEDTSVYYCAAEVRNGVFGKWNHYWGQGTQVTVSSGGGG SGGGSEVQLVESGGGLVQAGESLTLSCAASGRTLSAYIMGWFRQAPGKEREFVAGIWSGGYTHL ADSAKGRFSISRDNAKNTVYLQMNGLKPEDTAVYYCAAGLRGRQYSNWGQGTQVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 08A5-9GS-08A10 82EVQLVESGGGLVQAGDSLRLSCAASGLTFSNY DMGWFRQAPGKEREFVGASWWSGGAPYYSDSVKGRFTISRDNAKNTVYLQANSLRPEDTAVY YCAAKRLRSFASGGSYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAS GSIFSIAAMGWYRQATGKQRELVATITDGGTTTYADSVKGRVTISRDRSANTVYLAMNNLKPDD TAVYYCYAYLRYTSRVPGDNYWGQGTQVTVS S08A10-9GS-Alb8- 83 EVQLVESGGGLVQAGGSLRLSCAASGSIFSIAA 9GS-08A10MGWYRQATGKQRELVATITDGGTTTYADSVK GRVTISRDRSANTVYLAMNNLKPDDTAVYYCYAYLRYTSRVPGDNYWGQGTQVTVSSGGGGS GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQAGGSLRLSCAASGSIFSIAAMGWYRQATGKQRELVATITDGGTTTYADSVKG RVTISRDRSANTVYLAMNNLKPDDTAVYYCYAYLRYTSRVPGDNYWGQGTQVTVSS 08A10-9GS- 84EVQLVESGGGLVQAGGSLRLSCAASGSIFSIAA 08A10-9GS-Alb8MGWYRQATGKQRELVATITDGGTTTYADSVK GRVTISRDRSANTVYLAMNNLKPDDTAVYYCYAYLRYTSRVPGDNYWGQGTQVTVSSGGGGS GGGSEVQLVESGGGLVQAGGSLRLSCAASGSIFSIAAMGWYRQATGKQRELVATITDGGTTTYA DSVKGRVTISRDRSANTVYLAMNNLKPDDTAVYYCYAYLRYTSRVPGDNYWGQGTQVTVSSGG GGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSD TLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 08A5-9GS-08A10- 85EVQLVESGGGLVQAGDSLRLSCAASGLTFSNY 9GS-Alb8 DMGWFRQAPGKEREFVGASWWSGGAPYYSDSVKGRFTISRDNAKNTVYLQANSLRPEDTAVY YCAAKRLRSFASGGSYDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGSLRLSCAAS GSIFSIAAMGWYRQATGKQRELVATITDGGTTTYADSVKGRVTISRDRSANTVYLAMNNLKPDD TAVYYCYAYLRYTSRVPGDNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSC AASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSL RPEDTAVYYCTIGGSLSRSSQGTLVTVSS07B11-9GS- 86 EVQLVESGGNLVQAGGSLGLSCAASVSISSIHI 238D2 (238D2MGWYRQAPGKQRDLVATITSGGSTAYADSVK is directedGRFTVSKDNAKNTVYLQMDSLKPEDTSVYYC against AAEVRNGVFGKWNHYWGQGTQVTVSSGGGGCXCR4) SGGGSEVQLVESGGGLVQTGGSLRLSCAASGF TFSSYAMSWVRQAPGKGLEWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNMLYLQMYSLKPEDT AVYYCAKSRVSRTGLYTYDNRGQGTQVTVSS07C3-9GS-238D4 87 EVQLVESGGGLVQAGESLTLSCAASGRTLSAYI (238D4 isMGWFRQAPGKEREFVAGIWSGGYTHLADSAK directedGRFSISRDNAKNTVYLQMNGLKPEDTAVYYCA against AGLRGRQYSNWGQGTQVTVSSGGGGSGGGSECXCR4) VQLMESGGGLVQAGGSLRLSCAASGRTFNNY AMGWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTA VYTCAASAIGSGALRRFEYDYSGQGTQVTVSS08A10-9GS-Alb8- 88 EVQLVESGGGLVQAGGSLRLSCAASGSIFSIAA 9GS-238D2MGWYRQATGKQRELVATITDGGTTTYADSVK (238D2 isGRVTISRDRSANTVYLAMNNLKPDDTAVYYC directed YAYLRYTSRVPGDNYWGQGTQVTVSSGGGGSagainst GGGSEVQLVESGGGLVQPGNSLRLSCAASGFT CXCR4)FSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY ADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQTGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIKSSGDSTRYAGSV KGRFTISRDNAKNMLYLQMYSLKPEDTAVYYCAKSRVSRTGLYTYDNRGQGTQVTVSS 08A5-9GS- 89EVQLVESGGGLVQAGDSLRLSCAASGLTFSNY 238D4-9GS-Alb8DMGWFRQAPGKEREFVGASWWSGGAPYYSD (238D4 isSVKGRFTISRDNAKNTVYLQANSLRPEDTAVY directedYCAAKRLRSFASGGSYDYWGQGTQVTVSSGG against GGSGGGSEVQLMESGGGLVQAGGSLRLSCAASCXCR4) GRTFNNYAMGWFRRAPGKEREFVAAITRSGVR SGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQG TQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWV SSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVS S

TABLE B-5 Linker sequences of the invention Name of SEQ linker ID NO:Amino acid sequences 5GS 49 GGGGS 6GS 50 SGGSGGS 9GS 51 GGGGSGGGS 10GS52 GGGGSGGGGS 15GS 53 GGGGSGGGGSGGGGS 18GS 54 GGGGSGGGGSGGGGGGGS 20GS 55GGGGSGGGGSGGGGSGGGGS 25GS 56 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS 57GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS 58 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

TABLE B-6 Nucleic acid sequences of the invention SEQ ID Name of cloneNO: Nucleic acid sequences 07B11 59 GAGGTGCAATTGGTGGAGTCTGGGGGAAACTTGGTGCAGGCTGGGGGGTCTCTGGGACTCTC CTGTGCAGCCTCTGTAAGCATCTCCAGTATCCATATCATGGGCTGGTACCGGCAGGCTCCAGG CAAACAGCGCGACTTGGTCGCTACTATTACTAGTGGTGGTAGCACAGCATATGCAGACTCCG TGAAGGGACGATTCACCGTCTCCAAAGACAACGCCAAGAACACGGTGTATCTGCAAATGGAC AGCCTGAAACCTGAGGACACATCCGTCTATTACTGTGCAGCCGAGGTCAGAAATGGGGTGTT TGGAAAATGGAATCACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA 07C3 60 GAGGTGCAATTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGAGTCTCTGACTCTCTC CTGTGCAGCCTCTGGACGCACCTTAAGTGCCTATATCATGGGCTGGTTCCGCCAGGCTCCAG GGAAGGAGCGGGAGTTTGTAGCCGGTATCTGGAGTGGTGGTTACACACACCTTGCAGACTCC GCGAAGGGCCGATTCAGCATCTCTAGAGACAACGCCAAGAACACTGTATATCTGCAAATGAA CGGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTGCAGCAGGTCTGAGAGGCCGCCAGT ATAGTAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA 08A5 61 GAGGTGCAATTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTC CTGTGCAGCCTCTGGACTCACTTTCAGTAACTATGACATGGGCTGGTTCCGCCAGGCTCCAGG GAAGGAGCGTGAATTTGTAGGGGCTAGTTGGTGGAGTGGTGGTGCCCCATACTATTCAGACT CCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAGCG AACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCCAAAAGGCTGCGTAGTTT CGCCTCCGGTGGGTCGTATGATTACTGGGGTCAGGGGACCCAGGTCACCGTCTCCTCA 08A10 62 GAGTCTGGGGGAGGCTTGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCTTCTGGA AGCATCTTCAGTATCGCTGCCATGGGCTGGTACCGCCAGGCTACAGGGAAGCAGCGCGAGT TGGTCGCAACTATCACTGATGGCGGTACGACAACCTATGCAGACTCCGTGAAGGGCCGAGTC ACCATCTCCAGGGACAGGTCTGCGAACACGGTGTATCTGGCAATGAACAATTTGAAACCTGA TGACACAGCCGTCTATTATTGTTATGCGTATCTGCGCTATACAAGCAGAGTACCTGGCGATAA CTACTGGGGCCAGGGGACCCAGGTCACCGTC TCCTCA14G3 63 GAGGTGCAATTGGTGGAGTCTGGGGGAGGCT TGGTGCAGCCTGGGGGGTCTCTGAGAATTTCCTGTGCAGCCTCTGGAAGCATCTACCTTATC AATTACATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAACGCTTAC TAGTGGTGGTAGTACCAACTATGCAGGCTCCGTGAAGGGCCGATTCGCCATCTCCAGAGACA ACGCCAAGAACACGGTTTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTAT TACTGTAATATAGGAGGAACGCTATACGACAGAAGGCGGTTTGAATCCTGGGGCCAGGGGAC CCAGGTCACCGTCTCCTCAG 07B11-9GS-Alb8 73GAGGTGCAATTGGTGGAGTCTGGGGGAAACT TGGTGCAGGCTGGGGGGTCTCTGGGACTCTCCTGTGCAGCCTCTGTAAGCATCTCCAGTATCC ATATCATGGGCTGGTACCGGCAGGCTCCAGGCAAACAGCGCGACTTGGTCGCTACTATTACT AGTGGTGGTAGCACAGCATATGCAGACTCCGTGAAGGGACGATTCACCGTCTCCAAAGACAA CGCCAAGAACACGGTGTATCTGCAAATGGACAGCCTGAAACCTGAGGACACATCCGTCTATT ACTGTGCAGCCGAGGTCAGAAATGGGGTGTTTGGAAAATGGAATCACTACTGGGGCCAGGG GACCCAGGTCACGGTCTCCTCAGGAGGTGGCGGGTCCGGAGGCGGATCCGAGGTACAGCTG GTGGAGTCTGGGGGTGGCTTGGTGCAACCGGGTAACAGTCTGCGCCTTAGCTGCGCAGCGTC TGGCTTTACCTTCAGCTCCTTTGGCATGAGCTGGGTTCGCCAGGCTCCGGGAAAAGGACTGG AATGGGTTTCGTCTATTAGCGGCAGTGGTAGCGATACGCTCTACGCGGACTCCGTGAAGGGC CGTTTCACCATCTCCCGCGATAACGCCAAAACTACACTGTATCTGCAAATGAATAGCCTGCG TCCTGAAGACACGGCCGTTTATTACTGTACTATTGGTGGCTCGTTAAGCCGTTCTTCACAGG GTACCCTGGTCACCGTCTCCTCA 07C3-9GS-Alb8 74GAGGTGCAATTGGTGGAGTCTGGGGGAGGAT TGGTGCAGGCTGGGGAGTCTCTGACTCTCTCCTGTGCAGCCTCTGGACGCACCTTAAGTGCC TATATCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGGGAGTTTGTAGCCGGTATCTG GAGTGGTGGTTACACACACCTTGCAGACTCCGCGAAGGGCCGATTCAGCATCTCTAGAGACA ACGCCAAGAACACTGTATATCTGCAAATGAACGGCCTGAAACCTGAGGACACGGCCGTCTAT TACTGTGCAGCAGGTCTGAGAGGCCGCCAGTATAGTAACTGGGGCCAGGGGACCCAGGTCAC GGTCTCCTCAGGAGGTGGCGGGTCCGGAGGCGGATCCGAGGTACAGCTGGTGGAGTCTGGGG GTGGCTTGGTGCAACCGGGTAACAGTCTGCGCCTTAGCTGCGCAGCGTCTGGCTTTACCTTCA GCTCCTTTGGCATGAGCTGGGTTCGCCAGGCTCCGGGAAAAGGACTGGAATGGGTTTCGTCT ATTAGCGGCAGTGGTAGCGATACGCTCTACGCGGACTCCGTGAAGGGCCGTTTCACCATCTC CCGCGATAACGCCAAAACTACACTGTATCTGCAAATGAATAGCCTGCGTCCTGAAGACACGG CCGTTTATTACTGTACTATTGGTGGCTCGTTAAGCCGTTCTTCACAGGGTACCCTGGTCACCG TCTCCTCA 08A5-9GS-Alb8 75GAGGTGCAATTGGTGGAGTCTGGGGGAGGAT TGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGGACTCACTTTCAGTAACT ATGACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAATTTGTAGGGGCTAGTTGG TGGAGTGGTGGTGCCCCATACTATTCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGA CAACGCCAAGAACACGGTGTATCTGCAAGCGAACAGCCTGAGACCTGAGGACACGGCCGTTT ATTACTGTGCAGCCAAAAGGCTGCGTAGTTTCGCCTCCGGTGGGTCGTATGATTACTGGGGT CAGGGGACCCAGGTCACGGTCTCCTCAGGAGGTGGCGGGTCCGGAGGCGGATCCGAGGTAC AGCTGGTGGAGTCTGGGGGTGGCTTGGTGCAACCGGGTAACAGTCTGCGCCTTAGCTGCGCA GCGTCTGGCTTTACCTTCAGCTCCTTTGGCATGAGCTGGGTTCGCCAGGCTCCGGGAAAAGG ACTGGAATGGGTTTCGTCTATTAGCGGCAGTGGTAGCGATACGCTCTACGCGGACTCCGTGA AGGGCCGTTTCACCATCTCCCGCGATAACGCCAAAACTACACTGTATCTGCAAATGAATAGC CTGCGTCCTGAAGACACGGCCGTTTATTACTGTACTATTGGTGGCTCGTTAAGCCGTTCTTCA CAGGGTACCCTGGTCACCGTCTCCTCA08A10-9GS-Alb8 76 GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGAGGGTCTCTGAGACTCTC CTGTGCAGCTTCTGGAAGCATCTTCAGTATCGCTGCCATGGGCTGGTACCGCCAGGCTACAG GGAAGCAGCGCGAGTTGGTCGCAACTATCACTGATGGCGGTACGACAACCTATGCAGACTCC GTGAAGGGCCGAGTCACCATCTCCAGGGACAGGTCTGCGAACACGGTGTATCTGGCAATGAA CAATTTGAAACCTGATGACACAGCCGTCTATTATTGTTATGCGTATCTGCGCTATACAAGCA GAGTACCTGGCGATAACTACTGGGGCCAGGGGACCCAGGTCACGGTCTCCTCAGGAGGTGGC GGGTCCGGAGGCGGATCCGAGGTACAGCTGGTGGAGTCTGGGGGTGGCTTGGTGCAACCGG GTAACAGTCTGCGCCTTAGCTGCGCAGCGTCTGGCTTTACCTTCAGCTCCTTTGGCATGAGCT GGGTTCGCCAGGCTCCGGGAAAAGGACTGGAATGGGTTTCGTCTATTAGCGGCAGTGGTAG CGATACGCTCTACGCGGACTCCGTGAAGGGCCGTTTCACCATCTCCCGCGATAACGCCAAAA CTACACTGTATCTGCAAATGAATAGCCTGCGTCCTGAAGACACGGCCGTTTATTACTGTACT ATTGGTGGCTCGTTAAGCCGTTCTTCACAGGGTACCCTGGTCACCGTCTCCTCA 14G3-9GS-Alb8 77 GAGGTGCAATTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGAATTTC CTGTGCAGCCTCTGGAAGCATCTACCTTATCAATTACATGGGCTGGTACCGCCAGGCTCCAG GGAAGCAGCGCGAGTTGGTCGCAACGCTTACTAGTGGTGGTAGTACCAACTATGCAGGCTCC GTGAAGGGCCGATTCGCCATCTCCAGAGACAACGCCAAGAACACGGTTTATCTGCAAATGAA CAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAATATAGGAGGAACGCTATACGACA GAAGGCGGTTTGAATCCTGGGGCCAGGGGACCCAGGTCACGGTCTCCTCAGGAGGTGGCGGG TCCGGAGGCGGATCCGAGGTACAGCTGGTGGAGTCTGGGGGTGGCTTGGTGCAACCGGGTAA CAGTCTGCGCCTTAGCTGCGCAGCGTCTGGCTTTACCTTCAGCTCCTTTGGCATGAGCTGGGT TCGCCAGGCTCCGGGAAAAGGACTGGAATGGGTTTCGTCTATTAGCGGCAGTGGTAGCGAT ACGCTCTACGCGGACTCCGTGAAGGGCCGTTTCACCATCTCCCGCGATAACGCCAAAACTAC ACTGTATCTGCAAATGAATAGCCTGCGTCCTGAAGACACGGCCGTTTATTACTGTACTATTG GTGGCTCGTTAAGCCGTTCTTCACAGGGTACCCTGGTCACCGTCTCCTCA

Example 1 Cloning

Human CXCR7 (hCXCR7), mouse CXCR7 (Open Biosystems) and cynomolgusencoding cDNA (Table B-1) were cloned into pVAX-1 (Invitrogen) and/orpcDNA3.1 (Invitrogen). Transfection of pVAX1-hCXCR7 andpcDNA3.1-human(mouse)(cyno)CXCR7 constructs in Hek293 cells resulted inCXCR7 cell surface expression as shown by FACS analysis using the humanCXCR7 specific monoclonal antibody 11G8 (R&D Systems) and a PE-labeledgoat anti-mouse IgG detecting antibody (Jackson ImmunoResearch Inc.).

Example 2 Immunizations

For genetic immunization, endotoxin-free pVAX1-CXCR7 plasmid wasproduced, dissolved to a concentration of 2 mg/mL in 0.9% saline andstored at −20° C. Four llamas (391, 395, 396 and 397) were immunizedwith 2 mg pVAX1-hCXCR7 via intradermal Jet injection (Akra DermoJetFrance) for four times with two weeks intervals. Three weeks after thefinal DNA immunization, the 4 animals received a boost with camel kidney(CAKI) cells (Nguyen et al. 2001. Adv. Immunol. 79: 261-296) (2×10⁷cells) stably expressing hCXCR7. Three llamas (385, 387 and 404) wereimmunized with four injections of 2×10⁷ HEK293 cells transfected withpcDNA3.1-hCXCR7 with two weeks intervals. From llamas 391, 395, 396 and397, peripheral blood lymphocytes were collected 4 days and 10 daysafter the last DNA immunization and 3 days and 9 days after the cellboost. From llamas 385, 387 and 404, peripheral blood lymphocytes werecollected 4 and 8 days after the final cell injection. Additionally, abiopsy of the palpable bow lymph node (LN) was collected from each llamavia local surgery 3 days after the last cell boost. From all lymphocyteharboring immune tissues total RNA was extracted and used as template toprepare cDNA.

Example 3 Library Construction

Libraries were constructed from immune tissues collected from allllamas. In short, cDNA was prepared from the extracted total RNA samples(example 2) and used to amplify the cDNA repertoire via nested PCR aspreviously described (WO 02/085945 and WO 04/049794). The PCR productswere digested with SfiI (introduced via nested PCR in the FR1 primerregion) and BstEII (restriction site naturally occurring in FR4) andfollowing gel electrophoresis, the DNA fragment of approximately 400 bpswas purified from gel. The amplified cDNA repertoire was ligated intothe corresponding restriction sites of SfiI-BstEII digested phagedisplay vector (pAX50) to obtain a library after electroporation ofEscherichia coli TG1. This display vector allows the production of phageparticles, expressing the individual VHHs (hereinforth also referred toas Nanobodies) as a fusion protein with a C-terminal Myc-His6-tag(hereinforth also TAG-1 or SEQ ID NO: 71) and with the geneIII product.

Libraries were rescued by growing the bacteria to logarithmic phase(OD₆₀₀=0.5), followed by infection with helper phage to obtainrecombinant phage expressing the cloned Nanobodies on tip of the phageas a pIII fusion protein. Phage was stored after filter sterilization at4° C. for further use.

Example 4 Selections of Phage Displaying Human CXCR7 Binding Nanobodies

Phage from the above libraries were used for selections on hCXCR7virus-like particles (VLP; Integral Molecular), intact CXCR7 expressingcells, membrane extracts from CXCR7 expressing cells and peptides.

In a first selection round, 10 units of VLPs derived from hCXCR7transfected HEK293 cells were coated in 96-well MAXISORP™ plate (NUNC®)and blocked with low-fat milk powder (Marvell 4% in PBS). After 2 hoursof incubation with rescued phage, trypsin elution (1 mg/ml) was allowedfor 15 minutes at room temperature subsequent to 20 PBS washes. Proteaseactivity was immediately neutralized by applying 16 mM proteaseinhibitor ABSF. The round 1 phage outputs were rescued and a secondselection round was performed on 10 or 1 units of plate-immobilizedhCXCR7 VLPs. The round 2 phage outputs selected on 10 or 1 units plateimmobilized hCXCR7 VLPs were infected into TG1 cells and plated on agarplates (LB+Amp+2% glucose).

Individual colonies of E. coli TG1 infected with the eluted phage poolsobtained after selections were picked up and grown in 96-deep-wellplates to produce monoclonal phage after addition of helper phage. Theproduction of monoclonal Nanobodies was induced by the addition ofisopropyl-b-D-thiogalactopyranoside (IPTG). The perisplasmic fractioncontaining Nanobodies was then prepared by freezing-thawing of thebacterial pellet in PBS and subsequent centrifugation to remove cellfragments.

Example 5 Identification of CXCR7 Specific Nanobodies by Phage ELISA

From all round 2 selection outputs clones were screened in phage ELISAon 2 units of immobilized CXCR7 VLPs applying 10-fold dilutions of phagesupernatant. After incubation with HRP-conjugated monoclonal-anti-M13antibody (GE, Cat#363761) and several washings, phage binding wasrevealed using TMB substrate (Pierce). The reaction was stopped withH₂SO₄ and the absorbance was measured at 450 nm using Sunrise TECANspectrophotometer (TECAN). Nanobodies, showing a minimally 2-foldincreased ELISA signal on hCXCR7 VLPs over non-transfected control VLPs,were considered to be CXCR7 specific. CXCR7 specific Nanobodies weresequenced and redundant Nanobodies (identical AA sequence) were removed.This resulted in the identification of 78 unique sequences, belonging to45 distinct Nanobody B-cell lineages. Phage ELISA data forrepresentative clones from distinct Nanobody B-cell lineages arerepresented in Table B-7 and indicate that the Nanobodies do bind tohuman CXCR7 on VLP.

TABLE B-7 CXCR7 screening results-ELISA. CXCR7-LP LP Null-LP FoldCXCR7-LP/ Clones with Tag-1 2U/well [OD] [OD] Null-LP 08A5 0.019 0.0082.4 08A10 0.104 0.006 17.3 14G3 0.316 0.043 7.3 07B11 0.041 0.010 4.107C3 0.053 0.012 4.4

Example 6 Identification of CXCR7 Specific Nanobodies by FACS Analysis

Clones representing distinct Nanobody B-cell lineages were tested asperiplasmic extracts for their binding to cell surface exposed CXCR7. Inthis assay, 5-fold dilutions of periplasmic extract were incubated withHek293 hCXCR7 and Hek293 wt cells. Binding of the Nanobodies wasdetected using mouse anti-myc (Serotec), followed by anti-mouse IgG-PE(Jackson Immununoresearch). Binding signals of selected Nanobody clones(mcf values and ratios of binding) are represented in Table B-8 andindicate that the Nanobodies do bind to cellular human CXCR7.

TABLE B-8 CXCR7 screening results-FACS analysis. Clones with Hek-CXCR7Hek wt Fold Hek Tag-1 Family Llama [MCF] [MCF] CXCR7/CXCR4 08A5 14 39618621 310 60.1 08A10 20 397 27411 322 85.1 14G3 23 385 45811 381 120.207B11 34 395 42877 389 110.2 07C3 37 391 23359 319 73.2

Example 7 Expression of CXCR7 Specific Nanobodies

Selected Nanobodies were recloned in E. coli expression vector pAX100and expressed as C-terminal linked myc, His6 (hereforth also Tag-2 orSEQ ID NO: 72)-tagged proteins. Nanobodies were also expressed as fusionproteins comprising Alb8 (Nanobody-linker-Alb8-myc-His6) (see sequencesSEQ ID NO: 44 to 48—Table B-4) or as tagless Nanobodies. Expression wasinduced by IPTG and allowed to continue for 4 h at 37° C. After spinningthe cell cultures, periplasmic extracts were prepared by freeze-thawingthe pellets. Nanobodies were purified from these extracts usingimmobilized metal affinity chromatography (IMAC) and a buffer exchangeto D-PBS.

Example 8 Binding FACS Analysis of CXCR7 Specific Nanobodies

Serial dilutions of purified proteins (concentration range: 400 nM-180pM) were incubated with stable HEK-CXCR7 cells for 30 min at 4° C. andbinding was detected using anti-mouse anti-myc (Serotec) and anti-mouseIgG-PE (Jackson Immunoresearch). The half maximal effectiveconcentration (EC50) values and upper plateau levels of selected clonesare depicted in Table B-9. These data confirm the screening data andunderscore that the indicated Nanobodies bind to cellular human CXCR7.

TABLE B-9 Binding FACS analysis Clones with Tag-2 EC50 Plateau [mcf]08A5 8.9 28474 08A10 11.9 34896 14G3 10.2 23807 07B11 30.5 24898 07C33.3 33113

Example 9 Nanobodies Compete with SDF-1 for CXCR7 Binding (DisplacementAssay)

In order to assess the competition capacity, Nanobodies were evaluatedin SDF1 ligand displacement assays using stable NIH3T3-hCXCR7 cells. 24h after seeding the cells, the cells were pre-incubated for 1 h at 4° C.with a dilution series of purified monovalent Nanobodies and thecorresponding C-terminal Tag-2 tagged fusion proteins to the human serumalbumin binding Nanobody Alb8 (see Table B-4: SEQ ID NOs: 44 to 48wherein the polypeptides are all C-terminal tagged with Tag-2). Alsoreference molecules Mab 8F11 (Biolegend), Mab 11G8 (R&D) and unlabelledSDF1 were included in the assay. Radiolabeled [¹²⁵I]-CXCL12 was dilutedand added to the cells to reach a final concentration of 75 pM and cellswere incubated for 3 h at 4° C. After incubation, cells were washedtwice, lysed with RIPA buffer and the ¹²⁵I signal was measured. AverageKi values and the percentage of displacement relative to thedisplacement of cold SDF1, are shown in Table B-10. The competition oftested Nanobodies and 8F11 Mab is between 73 and 83%, relative tocompetition with unlabelled SDF1. This level of displacement correspondto a 100% blocking of the CXCR7 protein, as the remaining SDF-1 bindingis believed not to be CXCR7 mediated, but due to the SDF-1 interactionwith heparan sulfate proteoglycans. Fusion to the human serumalbumin-binding Nanobody Alb8 has no significant effect on Ki values.

TABLE B-10 Displacement assay Average Ki dis- SEM SDF-1 Clones withwhole 3T3 place- SEM displacement Tag-2 [nM] ment n Ki (%) 08A5 13.6 778 2.5 6.4 08A5-9GS-Alb8 17.9 1 08A10 12.1 75 8 1.8 3.3 08A10-9GS-Alb814.1 1 14G3 3.0 73 6 0.6 3.3 14G3-9GS-Alb8 3.5 1 07B11 96.1 75 2 1.3 1.507B11-9GS-Alb8 82.4 1 07C3 12.2 78 2 6.6 15.0  10.2 1 SDF1 0.121 100 15  0.019 0.0 11G8 Mab 4.4 24 3 2.7 2.0 8F11 Mab 5.9 73 6 2.4 4.1

Example 10 Nanobodies Compete with SDF-1 for CXCR7 Binding (FACS Assay)

The potency of Nanobody 7CXCR07C3 and Mab 8F11 (Biolegend) to competewith SDF1 was evaluated in competition FACS with HEK-hCXCR7 cells. Cellswere incubated simultaneously with 4 nM biotinylated SDF1 (R&D) and withdiluted test molecules, for 2 h at 4° C. Binding of biotinylated SDF1was detected using streptavidin-PE. Competition curves are depicted inFIG. 1. In this assay, Mab8F11 and 07C3 competition is complete (>95%),relative to competition with unlabelled SDF1, underscoring the completeinhibition of the SDF-1-CXCR7 interaction.

Example 11 Nanobodies Compete with 11G8 Mab for CXCR7 Binding (FACSAssay)

The minimal epitope of Mab11G8 is known to be F₁₄SDISWP₂₀ located at theCXCR7 N-terminus (see e.g. WO2008048519). Competition of Nanobodies, Mab8F11 (Biolegend), Mab 11G8 (R&D) and Mab9C4 (MBL) with Mab11G8-APC wastested in FACS analysis. Cells were incubated simultaneously with 20 nMMab11G8-APC (R&D) and with diluted test molecules for 2 h at 4° C.Competition curves are depicted in FIG. 2. The level of competition withMab11G8-APC ranges from ˜20 to 100% suggesting that the respectiveNanobody epitopes match to a high degree (high % of competition) withthe 11G8 epitope or to a low degree (low % of competition) or induceallosteric changes affecting the 11G8 binding. This data indicates thatthe selected Nanobodies bind to divergent epitopes.

Example 12 Mouse/Cyno Cross-Reactivity

Taking into account CXCR7 extracellular domains Met₁-Lys₄₀,Ser₁₀₃-Lys₁₁₈, Lys₁₈₃-Glu₂₁₃ and Leu₂₇₃-Ala₂₉₆, only 9 residues (Leu₃,Ser₉, Phe₁₄, Ser₁₈, Met₃₃, Asn₃₆, Arg₂₈₈, His₂₉₁ and Ala₂₉₂) differbetween human and mouse CXCR7. HEK293 cells transfected respectivelywith pcDNA3.1-hCXCR7 and pcDNA3.1-mCXCR7 were used to testcross-reactive binding of Nanobodies to mCXCR7 in FACS analysis. Cellswere incubated with 32 nM Mab 11G8 (R&D), Mab 9C4 (MBL), Mab 8F11(Biolegend) or with 800 nM Nanobody for 2 h at 4° C. Nanobody bindingwas detected using mouse anti-myc (Serotec) and anti-mouse IgG-PE(Jackson Immunoresearch) and Mab binding by goat anti-mouse IgG-PE(Jackson Immunoresearch). Nanobodies 08A10, 14G3, 07B11 and Mab9C4 arenot cross-reactive to mouse CXCR7, indicating that at least one of thenine divergent residues is crucial for the binding to human CXCR7.Nanobodies 08A5 and 07C3 partially cross-react with mouse CXCR7,indicating that at least one of the nine divergent residues is involvedin binding with human CXCR7. Mab8F11 and Mab11G8 are cross-reactive tomouse CXCR7, indicating that none of the nine divergent residues isinvolved in binding with human CXCR7. Together, this shows that allNanobody fingerprints differ from Mab8F11 and Mab11G8 fingerprints.

Cross-reactive binding to cynomolgus CXCR7 was assessed in the same way.Taking into account CXCR7 extracellular domains Met₁-Lys₄₀,Ser₁₀₃-Lys₁₁₈, Lys₁₈₃-Glu₂₁₃ and Leu₂₇₃-Ala₂₉₆, only one residue (Leu₅)differs between cynomolgus and mouse CXCR7. Not unexpectedly, Nanobodies08A10, 14G3, 707B11, 08A5, 07C3 and Mab9C4, Mab8F11 and Mab11G8 are allcross-reactive to cynomolgus CXCR7.

TABLE B-11 Cross-reactivity to mouse CXCR7 Clones with Mouse Cyno Tag-2Family Llama crossreactivity crossreactivity 08A5 14 396 Partial Yes08A10 20 397 No Yes 14G3 23 385 No Yes 07B11 34 395 No Yes 07C3 37 391Partial Yes 8F11 Mab Yes Yes 11G8 Mab Yes Yes 9C4 Mab No Yes

We claim:
 1. A VHH or dAb that comprises an amino acid sequence with theformula 1:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4  (1); wherein FR1 to FR4 refer toframework regions 1 to 4 and are framework regions of a VHH or dAb;wherein CDR1 is SEQ ID NO: 13; CDR2 is SEQ ID NO: 23; and CDR3 is SEQ IDNO: 33; wherein said VHH or dAb specifically binds to human CXCR7. 2.The VHH or dAb according to claim 1, wherein the framework regions (FRs)have a sequence identity of more than 80% with the FRs of SEQ ID NOs: 8(FR1), 18 (FR2), 28 (FR3), and 38 (FR4).
 3. A polypeptide comprising theVHH or dAb of claim
 1. 4. The polypeptide according to claim 3, whereinthe VHH or dAb has a sequence identity of more than 80% with the VHH ordAb of SEQ ID NO:43.
 5. The polypeptide according to claim 3additionally comprising at least one human serum albumin binding VHH ordAb.
 6. The polypeptide according to claim 5, wherein said human serumalbumin binding VHH or dAb is Alb8 having SEQ ID NO:
 2. 7. Thepolypeptide according to claim 6, wherein the polypeptide has an aminoacid sequence with a sequence identity of more than 80% with thepolypeptide of SEQ ID NO:48.
 8. A pharmaceutical composition comprisingthe VHH or dAb according to claim 1 and a pharmaceutically acceptableexcipient.
 9. The VHH or dAb according to claim 1 for use in treatingcancer and/or inflammatory diseases.
 10. The VHH or dAb according toclaim 1 for use in treating rheumatoid arthritis.
 11. The VHH or dAbaccording to claim 1 for use in treating multiple sclerosis.
 12. Thepolypeptide according to claim 5, wherein said VHH or dAb and at leastone of the human serum albumin binding VHH or dAb are linked via alinker, said linker being selected from the group of linkers with SEQ IDNOs: 49 to
 58. 13. The polypeptide according to claim 6, wherein saidVHH or dAb and at least one of said Alb8 VHH or dAb having SEQ ID NO: 2are linked via a linker, said linker being selected from the group oflinkers with SEQ ID NOs: 49 to
 58. 14. A pharmaceutical compositioncomprising the polypeptide according to claim 3 and a pharmaceuticallyacceptable excipient.